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Sample records for affinity ic50 values

  1. The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments.

    PubMed

    Haupt, Lois J; Kazmi, Faraz; Ogilvie, Brian W; Buckley, David B; Smith, Brian D; Leatherman, Sarah; Paris, Brandy; Parkinson, Oliver; Parkinson, Andrew

    2015-11-01

    In the present study, we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of Ki for reversible cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC₅₀ values by two, based on the relationship (for competitive inhibition) in which Ki = IC₅₀/2 when [S] (substrate concentration) = Km (Michaelis-Menten constant). Values of Ki and IC₅₀ were determined under the following conditions: 1) the concentration of P450 marker substrate, [S], was equal to Km (for IC₅₀ determinations) and spanned Km (for Ki determinations); 2) the substrate incubation time was short (5 minutes) to minimize metabolism-dependent inhibition and inhibitor depletion; and 3) the concentration of human liver microsomes was low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted Ki values, based on IC₅₀/2, correlated strongly with experimentally observed Ki determinations [r = 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki values, and only one value fell outside a 3-fold range. In the case of noncompetitive inhibitors, Ki values predicted from IC₅₀/2 values were overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, Ki = IC₅₀ (not IC₅₀/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to Km, values of Ki for direct, reversible inhibition can be reliably estimated from values of IC₅₀/2. PMID:26354951

  2. The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments.

    PubMed

    Haupt, Lois J; Kazmi, Faraz; Ogilvie, Brian W; Buckley, David B; Smith, Brian D; Leatherman, Sarah; Paris, Brandy; Parkinson, Oliver; Parkinson, Andrew

    2015-11-01

    In the present study, we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of Ki for reversible cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC₅₀ values by two, based on the relationship (for competitive inhibition) in which Ki = IC₅₀/2 when [S] (substrate concentration) = Km (Michaelis-Menten constant). Values of Ki and IC₅₀ were determined under the following conditions: 1) the concentration of P450 marker substrate, [S], was equal to Km (for IC₅₀ determinations) and spanned Km (for Ki determinations); 2) the substrate incubation time was short (5 minutes) to minimize metabolism-dependent inhibition and inhibitor depletion; and 3) the concentration of human liver microsomes was low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted Ki values, based on IC₅₀/2, correlated strongly with experimentally observed Ki determinations [r = 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki values, and only one value fell outside a 3-fold range. In the case of noncompetitive inhibitors, Ki values predicted from IC₅₀/2 values were overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, Ki = IC₅₀ (not IC₅₀/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to Km, values of Ki for direct, reversible inhibition can be reliably estimated from values of IC₅₀/2.

  3. Determining the IC50 Values for Vorozole and Letrozole, on a Series of Human Liver Cytochrome P450s, to Help Determine the Binding Site of Vorozole in the Liver

    PubMed Central

    Raymond, Lendelle; Rayani, Nikita; Polson, Grace; Sikorski, Kylie; Lian, Ailin; VanAlstine-Parris, Melissa A.

    2015-01-01

    Vorozole and letrozole are third-generation aromatase (cytochrome P450 19A1) inhibitors. [11C]-Vorozole can be used as a radiotracer for aromatase in living animals but when administered by IV, it collects in the liver. Pretreatment with letrozole does not affect the binding of vorozole in the liver. In search of finding the protein responsible for the accumulation of vorozole in the liver, fluorometric high-throughput screening assays were used to test the inhibitory capability of vorozole and letrozole on a series of liver cytochrome P450s (CYP1A1, CYP1A2, CYP2A6, and CYP3A4). It was determined that vorozole is a potent inhibitor of CYP1A1 (IC50 = 0.469 μM) and a moderate inhibitor of CYP2A6 and CYP3A4 (IC50 = 24.4 and 98.1 μM, resp.). Letrozole is only a moderate inhibitor of CYP1A1 and CYP2A6 (IC50 = 69.8 and 106 μM) and a very weak inhibitor of CYP3A4 (<10% inhibition at 1 mM). Since CYP3A4 makes up the majority of the CYP content found in the human liver, and vorozole inhibits it moderately well but letrozole does not, CYP3A4 is a good candidate for the protein that [11C]-vorozole is binding to in the liver. PMID:26635974

  4. Development of Quantum Chemical Method to Calculate Half Maximal Inhibitory Concentration (IC50 ).

    PubMed

    Bag, Arijit; Ghorai, Pradip Kr

    2016-05-01

    Till date theoretical calculation of the half maximal inhibitory concentration (IC50 ) of a compound is based on different Quantitative Structure Activity Relationship (QSAR) models which are empirical methods. By using the Cheng-Prusoff equation it may be possible to compute IC50 , but this will be computationally very expensive as it requires explicit calculation of binding free energy of an inhibitor with respective protein or enzyme. In this article, for the first time we report an ab initio method to compute IC50 of a compound based only on the inhibitor itself where the effect of the protein is reflected through a proportionality constant. By using basic enzyme inhibition kinetics and thermodynamic relations, we derive an expression of IC50 in terms of hydrophobicity, electric dipole moment (μ) and reactivity descriptor (ω) of an inhibitor. We implement this theory to compute IC50 of 15 HIV-1 capsid inhibitors and compared them with experimental results and available other QASR based empirical results. Calculated values using our method are in very good agreement with the experimental values compared to the values calculated using other methods. PMID:27492086

  5. AVP-IC50 Pred: Multiple machine learning techniques-based prediction of peptide antiviral activity in terms of half maximal inhibitory concentration (IC50).

    PubMed

    Qureshi, Abid; Tandon, Himani; Kumar, Manoj

    2015-11-01

    Peptide-based antiviral therapeutics has gradually paved their way into mainstream drug discovery research. Experimental determination of peptides' antiviral activity as expressed by their IC50 values involves a lot of effort. Therefore, we have developed "AVP-IC50 Pred," a regression-based algorithm to predict the antiviral activity in terms of IC50 values (μM). A total of 759 non-redundant peptides from AVPdb and HIPdb were divided into a training/test set having 683 peptides (T(683)) and a validation set with 76 independent peptides (V(76)) for evaluation. We utilized important peptide sequence features like amino-acid compositions, binary profile of N8-C8 residues, physicochemical properties and their hybrids. Four different machine learning techniques (MLTs) namely Support vector machine, Random Forest, Instance-based classifier, and K-Star were employed. During 10-fold cross validation, we achieved maximum Pearson correlation coefficients (PCCs) of 0.66, 0.64, 0.56, 0.55, respectively, for the above MLTs using the best combination of feature sets. All the predictive models also performed well on the independent validation dataset and achieved maximum PCCs of 0.74, 0.68, 0.59, 0.57, respectively, on the best combination of feature sets. The AVP-IC50 Pred web server is anticipated to assist the researchers working on antiviral therapeutics by enabling them to computationally screen many compounds and focus experimental validation on the most promising set of peptides, thus reducing cost and time efforts. The server is available at http://crdd.osdd.net/servers/ic50avp.

  6. A novel application of t-statistics to objectively assess the quality of IC50 fits for P-glycoprotein and other transporters.

    PubMed

    O'Connor, Michael; Lee, Caroline; Ellens, Harma; Bentz, Joe

    2015-02-01

    Current USFDA and EMA guidance for drug transporter interactions is dependent on IC50 measurements as these are utilized in determining whether a clinical interaction study is warranted. It is therefore important not only to standardize transport inhibition assay systems but also to develop uniform statistical criteria with associated probability statements for generation of robust IC50 values, which can be easily adopted across the industry. The current work provides a quantitative examination of critical factors affecting the quality of IC50 fits for P-gp inhibition through simulations of perfect data with randomly added error as commonly observed in the large data set collected by the P-gp IC50 initiative. The types of errors simulated were (1) variability in replicate measures of transport activity; (2) transformations of error-contaminated transport activity data prior to IC50 fitting (such as performed when determining an IC50 for inhibition of P-gp based on efflux ratio); and (3) the lack of well defined "no inhibition" and "complete inhibition" plateaus. The effect of the algorithm used in fitting the inhibition curve (e.g., two or three parameter fits) was also investigated. These simulations provide strong quantitative support for the recommendations provided in Bentz et al. (2013) for the determination of IC50 values for P-gp and demonstrate the adverse effect of data transformation prior to fitting. Furthermore, the simulations validate uniform statistical criteria for robust IC50 fits in general, which can be easily implemented across the industry. A calibration of the t-statistic is provided through calculation of confidence intervals associated with the t-statistic. PMID:25692007

  7. In vitro predictability of drug-drug interaction likelihood of P-glycoprotein-mediated efflux of dabigatran etexilate based on [I]2/IC50 threshold.

    PubMed

    Kishimoto, Wataru; Ishiguro, Naoki; Ludwig-Schwellinger, Eva; Ebner, Thomas; Schaefer, Olaf

    2014-02-01

    Dabigatran etexilate, an oral, reversible, competitive, and direct thrombin inhibitor, is an in vitro and in vivo substrate of P-glycoprotein (P-gp). Dabigatran etexilate was proposed as an in vivo probe substrate for intestinal P-gp inhibition in a recent guidance on drug-drug interactions (DDI) from the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). We conducted transcellular transport studies across Caco-2 cell monolayers with dabigatran etexilate in the presence of various P-gp inhibitors to examine how well in vitro IC50 data, in combination with mathematical equations provided by regulatory guidances, predict DDI likelihood. From a set of potential P-gp inhibitors, clarithromycin, cyclosporin A, itraconazole, ketoconazole, quinidine, and ritonavir inhibited P-gp-mediated transport of dabigatran etexilate over a concentration range that may hypothetically occur in the intestine. IC50 values of P-gp inhibitors for dabigatran etexilate transport were comparable to those of digoxin, a well established in vitro and in vivo P-gp substrate. However, IC50 values varied depending whether they were calculated from efflux ratios or permeability coefficients. Prediction of DDI likelihood of P-gp inhibitors using IC50 values, the hypothetical concentration of P-gp inhibitors, and the cut-off value recommended by both the FDA and EMA were in line with the DDI occurrence in clinical studies with dabigatran etexilate. However, it has to be kept in mind that validity of the cut-off criteria proposed by the FDA and EMA depends on in vitro experimental systems and the IC50-calculation methods that are employed, as IC50 values are substantially influenced by these factors.

  8. Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC50) test using WST-1 assay

    NASA Astrophysics Data System (ADS)

    Rosli, Nur Shafawati binti; Rahman, Azhar Abdul; Aziz, Azlan Abdul; Shamsuddin, Shaharum

    2015-04-01

    Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC50 values in WST-1 assays. The IC50 values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles.

  9. A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination.

    PubMed

    Yin, Liusong; Stern, Lawrence J

    2014-04-01

    HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.

  10. Neuronal in vitro activity is more sensitive to valproate than intracellular ATP: Considerations on conversion problems of IC50 in vitro data for animal replacement.

    PubMed

    Nissen, Matthias; Buehler, Sebastian M; Stubbe, Marco; Gimsa, Jan

    2016-06-01

    We investigated the effects of acute valproate (VPA) on mouse embryonic primary cortex cells (MEPCs). Intracellular ATP concentrations were compared with changes in the mean action potential (AP) frequencies of MEPC networks growing on microelectrode arrays. Our data implies biphasic reactions towards increasing VPA concentrations for both parameters. Intracellular ATP and mean AP frequencies increased around characteristic concentrations of 0.15 and 0.07mM to hormetic plateaus of approx. 120% and 160% of their controls, before fading around 17 and 1.7 mM, respectively. The biphasic in vitro behavior of the two parameters hinders a simple extraction of IC50 and Hillslope values. Different ways of data-fitting with single and double logistic functions are discussed. For a typical hormetic increase of 60% above control, IC50 and Hillslope were decreased by 37% and 15%, respectively. Despite these marginal effects at a logarithmic concentration scale, the hormetic and double logistic behavior of parameters may provide information on the mode of action of toxic compounds. Comparison of our values with the LD50 of mice, recalculated by normalization to body mass, suggests that a neurotoxic rather than a cytotoxic mechanism is killing the animals. The future use of cellular microsystems to replace animal experiments will motivate the development of new microsensors, as well as the consideration of newly accessible parameters in systems biology models. PMID:27091084

  11. Food and value motivation: Linking consumer affinities to different types of food products.

    PubMed

    de Boer, Joop; Schösler, Hanna

    2016-08-01

    This study uses the consumer affinity concept to examine the multiple motives that may shape consumers' relationships with food. The concept was applied in a study on four broad product types in the Netherlands, which cover a wide range of the market and may each appeal to consumers with different affinities towards foods. These product types may be denoted as 'conventional', 'efficient', 'gourmet' and 'pure'. A comparative analysis, based on Higgins' Regulatory Focus Theory, was performed to examine whether food-related value motivations could explain different consumer affinities for these product types. The affinities of consumers were measured by means of a non-verbal, visual presentation of four samples of food products in a nationwide survey (n = 742) among consumers who were all involved in food purchasing and/or cooking. The affinities found could be predicted fairly well from a number of self-descriptions relating to food and eating, which expressed different combinations of type of value motivation and involvement with food. The analysis demonstrated the contrasting role of high and low involvement as well as the potential complementarity of promotion- and prevention-focused value motivation. It is suggested that knowledge of the relationships between product types, consumer affinities and value motivation can help improve the effectiveness of interventions that seek to promote healthy and sustainable diets in developed countries. PMID:27046434

  12. Food and value motivation: Linking consumer affinities to different types of food products.

    PubMed

    de Boer, Joop; Schösler, Hanna

    2016-08-01

    This study uses the consumer affinity concept to examine the multiple motives that may shape consumers' relationships with food. The concept was applied in a study on four broad product types in the Netherlands, which cover a wide range of the market and may each appeal to consumers with different affinities towards foods. These product types may be denoted as 'conventional', 'efficient', 'gourmet' and 'pure'. A comparative analysis, based on Higgins' Regulatory Focus Theory, was performed to examine whether food-related value motivations could explain different consumer affinities for these product types. The affinities of consumers were measured by means of a non-verbal, visual presentation of four samples of food products in a nationwide survey (n = 742) among consumers who were all involved in food purchasing and/or cooking. The affinities found could be predicted fairly well from a number of self-descriptions relating to food and eating, which expressed different combinations of type of value motivation and involvement with food. The analysis demonstrated the contrasting role of high and low involvement as well as the potential complementarity of promotion- and prevention-focused value motivation. It is suggested that knowledge of the relationships between product types, consumer affinities and value motivation can help improve the effectiveness of interventions that seek to promote healthy and sustainable diets in developed countries.

  13. Dual Incorporation of the in vitro Data (IC50) and in vivo (Cmax) Data for the Prediction of Area Under the Curve (AUC) for Statins using Regression Models Developed for Either Pravastatin or Simvastatin.

    PubMed

    Srinivas, N R

    2016-08-01

    Linear regression models utilizing a single time point (Cmax) has been reported for pravastatin and simvastatin. A new model was developed for the prediction of AUC of statins that utilized the slopes of the above 2 models, with pharmacokinetic (Cmax) and a pharmacodynamic (IC50 value) components for the statins. The prediction of AUCs for various statins (pravastatin, atorvastatin, simvastatin and rosuvastatin) was carried out using the newly developed dual pharmacokinetic and pharmacodynamic model. Generally, the AUC predictions were contained within 0.5 to 2-fold difference of the observed AUC suggesting utility of the new models. The root mean square error predictions were<45% for the 2 models. On the basis of the present work, it is feasible to utilize both pharmacokinetic (Cmax) and pharmacodynamic (IC50) data for effectively predicting the AUC for statins. Such a new concept as described in the work may have utility in both drug discovery and development stages.

  14. Dual Incorporation of the in vitro Data (IC50) and in vivo (Cmax) Data for the Prediction of Area Under the Curve (AUC) for Statins using Regression Models Developed for Either Pravastatin or Simvastatin.

    PubMed

    Srinivas, N R

    2016-08-01

    Linear regression models utilizing a single time point (Cmax) has been reported for pravastatin and simvastatin. A new model was developed for the prediction of AUC of statins that utilized the slopes of the above 2 models, with pharmacokinetic (Cmax) and a pharmacodynamic (IC50 value) components for the statins. The prediction of AUCs for various statins (pravastatin, atorvastatin, simvastatin and rosuvastatin) was carried out using the newly developed dual pharmacokinetic and pharmacodynamic model. Generally, the AUC predictions were contained within 0.5 to 2-fold difference of the observed AUC suggesting utility of the new models. The root mean square error predictions were<45% for the 2 models. On the basis of the present work, it is feasible to utilize both pharmacokinetic (Cmax) and pharmacodynamic (IC50) data for effectively predicting the AUC for statins. Such a new concept as described in the work may have utility in both drug discovery and development stages. PMID:27144659

  15. Ligand Efficiency Outperforms pIC50 on Both 2D MLR and 3D CoMFA Models: A Case Study on AR Antagonists.

    PubMed

    Li, Jiazhong; Bai, Fang; Liu, Huanxiang; Gramatica, Paola

    2015-12-01

    The concept of ligand efficiency is defined as biological activity in each molecular size and is widely accepted throughout the drug design community. Among different LE indices, surface efficiency index (SEI) was reported to be the best one in support vector machine modeling, much better than the generally and traditionally used end-point pIC50. In this study, 2D multiple linear regression and 3D comparative molecular field analysis methods are employed to investigate the structure-activity relationships of a series of androgen receptor antagonists, using pIC50 and SEI as dependent variables to verify the influence of using different kinds of end-points. The obtained results suggest that SEI outperforms pIC50 on both MLR and CoMFA models with higher stability and predictive ability. After analyzing the characteristics of the two dependent variables SEI and pIC50, we deduce that the superiority of SEI maybe lie in that SEI could reflect the relationship between molecular structures and corresponding bioactivities, in nature, better than pIC50. This study indicates that SEI could be a more rational parameter to be optimized in the drug discovery process than pIC50.

  16. An activated sludge-based biosensor for rapid IC50 estimation and on-line toxicity monitoring.

    PubMed

    Kong, Z; Vanrolleghem, P A; Verstraete, W

    1993-01-01

    The RODTOX (Rapid Oxygen Demand and TOXicity tester), an activated sludge-based respirographic biosensor, is a device for on-line monitoring of the short-term biochemical oxygen demand (stBOD) and potential toxicity of incoming wastewater on the basis of on-line interpretation of respirograms resulting from pulse additions of either calibration substrate or sample. The principle of toxicity detection is based on the comparison of calibration respirograms before and after receiving a potential toxicant. In this paper, the results of the RODTOX as an on-line toxicity monitor are presented. In addition, a simple and fast procedure to estimate the IC50 of a toxicant has been developed, and its validity and good repeatability demonstrated. The performance of this procedure is compared with that of the Microtox test.

  17. Small-conductance Ca(2+)-activated K(+) channels: Heterogeneous affinity in rat brain structures and cognitive modulation by specific blockers.

    PubMed

    Mpari, Bedel; Sreng, Leam; Regaya, Imed; Mourre, Christiane

    2008-07-28

    Small-conductance calcium-activated potassium channels (K(Ca)2) generating the medium afterhyperpolarization seen after an action potential modulate the neuronal integration signal. The effects of two K(Ca)2 channel blockers, apamin, specific to K(Ca)2.2 and K(Ca)2.3 channels, and lei-Dab7, which binds to K(Ca)2.2 channels only, were compared to evaluate the involvement of K(Ca)2 channel subunits in behavior, spatial learning and memory in rats. Intracerebroventricular (9-5 ng) injections of lei-dab7 decreased locomotor activity, food intake and body weight in rats deprived of food. A dose of 3 ng lei-Dab7 had no effect on these types of behavior. We therefore used this dose for attention and memory tasks. No modification to attention or memory was observed in a spatial radial-arm maze task with rats given 3 ng lei-Dab7, whereas apamin (0.3 ng) improved reference memory and accelerated changes of strategy from egocentric to allocentric. These findings suggest that K(Ca)2.3 blockade improves memory in rats. Lei-Dab7 entirely outcompeted the binding of iodinated apamin to 64 brain structures (mean IC(50): 34.5 nM), although IC(50) values were highly variable. By contrast, overall IC(50) values for apamin were close to mean values (11.3 pM). The very low affinity of the hippocampus and neocortex for lei-Dab7 may account for the absence of a behavioral effect of this compound. The variability of IC(50) values suggests that K(Ca)2 channel composition varies considerably as a function of the brain structure considered. PMID:18561910

  18. Additive method for the prediction of protein-peptide binding affinity. Application to the MHC class I molecule HLA-A*0201.

    PubMed

    Doytchinova, Irini A; Blythe, Martin J; Flower, Darren R

    2002-01-01

    A method has been developed for prediction of binding affinities between proteins and peptides. We exemplify the method through its application to binding predictions of peptides with affinity to major histocompatibility complex class I molecule HLA-A*0201. The method is named "additive" because it is based on the assumption that the binding affinity of a peptide could be presented as a sum of the contributions of the amino acids at each position and the interactions between them. The amino acid contributions and the contributions of the interactions between adjacent side chains and every second side chain were derived using a partial least squares (PLS) statistical methodology using a training set of 420 experimental IC50 values. The predictive power of the method was assessed using rigorous cross-validation and using an independent test set of 89 peptides. The mean value of the residuals between the experimental and predicted pIC50 values was 0.508 for this test set. The additive method was implemented in a program for rapid T-cell epitope search. It is universal and can be applied to any peptide-protein interaction where binding data is known. PMID:12645903

  19. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M.

  20. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  1. Selection of three out of 24 anti-cancer agents in poorly-differentiated gastric cancer cell lines, evaluated by the AUC/delta IC50 ratio.

    PubMed

    Nozue, M; Nishida, M; Todoroki, T; Fukao, K; Tanaka, M

    1995-04-01

    The purpose of this study was to screen 24 anti-cancer drugs, either in use or in clinical study, using four cell lines, all of which originated from poorly-differentiated gastric cancers. The MTT assay was used at 1, 6, 24 or 72 h exposure times as the chemosensitivity test. We also examined P-glycoprotein expression, mdr-1 gene amplification and the modifier effect of verapamil. All four cell lines generally showed the same chemosensitivity pattern, while GCIY cells showed mdr-1 gene amplification and P-glycoprotein expression, and KATOIII cells showed the multidrug resistant pattern without P-glycoprotein expression. Both cell lines acquired higher chemosensitivity after verapamil addition. All IC50 data (with or without verapamil) were multiplied by exposure time (delta IC50) and compared with the clinical 'area under the concentration curve (AUC)'. SN-38 with/without verapamil, cisplatin with verapamil and pirarubicin with/without verapamil seemed to be the best candidates for poorly-differentiated gastric cancer chemotherapy. Plant alkaloids could also be candidates. With further experiments, we may be able to deduce commonly effective chemotherapy for poorly-differentiated gastric cancer from these drugs. PMID:7795277

  2. Synthesis of 24,24-Difluoro-1,3-cis-25-dihydroxy-19-norvitamin D3 Derivatives and Evaluation of Their Vitamin D Receptor-Binding Affinity.

    PubMed

    Biswas, Tanima; Akagi, Yusuke; Usuda, Kosuke; Yasui, Koji; Shimizu, Isao; Okamoto, Mayumi; Uesugi, Motonari; Hosokawa, Seijiro; Nagasawa, Kazuo

    2016-01-01

    Two vitamin D3 derivatives, namely 24,24-difluoro-1β,3β,25-dihydroxy-19-norvitamin D3 (6a) and 24,24-difluoro-1α,3α,25-dihydroxy-19-norvitamin D3 (6b), were synthesized via a convergent route employing Julia-Kocienski olefination as a key step. Compounds 6a and b bound to vitamin D receptor (VDR) with IC50 values of 64.8 and 57.6 nM, respectively, exhibiting about 400- and 30-fold greater binding affinity than the corresponding non-fluorinated derivatives 5a and b. PMID:27476947

  3. CORAL: prediction of binding affinity and efficacy of thyroid hormone receptor ligands.

    PubMed

    Toropova, A P; Toropov, A A; Benfenati, E

    2015-08-28

    Quantitative structure - activity relationships (QSARs) for binding affinity of thyroid hormone receptors based on attributes of molecular structure extracted from simplified molecular input-line entry systems (SMILES) are established using the CORAL software (http://www.insilico.eu/coral). The half maximal inhibitory concentration (IC50) is used as the measure of the binding affinity of thyroid hormone receptors. Molecular features which are statistically reliable promoters of increase and decrease for IC50 are suggested. The examples of modifications of molecular structure which lead to the increase or to the decrease of the endpoint are represented. PMID:26188619

  4. Bethe Ansatz and the Spectral Theory of Affine Lie algebra-Valued Connections II: The Non Simply-Laced Case

    NASA Astrophysics Data System (ADS)

    Masoero, Davide; Raimondo, Andrea; Valeri, Daniele

    2016-09-01

    We assess the ODE/IM correspondence for the quantum g -KdV model, for a non-simply laced Lie algebra g. This is done by studying a meromorphic connection with values in the Langlands dual algebra of the affine Lie algebra g^{(1)} , and constructing the relevant {Ψ} -system among subdominant solutions. We then use the {Ψ} -system to prove that the generalized spectral determinants satisfy the Bethe Ansatz equations of the quantum g -KdV model. We also consider generalized Airy functions for twisted Kac-Moody algebras and we construct new explicit solutions to the Bethe Ansatz equations. The paper is a continuation of our previous work on the ODE/IM correspondence for simply-laced Lie algebras.

  5. In vitro neuraminidase inhibitory concentration (IC50) of four neuraminidase inhibitors against clinical isolates of the influenza viruses circulating in the 2010-2011 to 2014-2015 Japanese influenza seasons.

    PubMed

    Ikematsu, Hideyuki; Kawai, Naoki; Iwaki, Norio; Kashiwagi, Seizaburo

    2016-09-01

    To assess the extent of viral resistance to the four neuraminidase inhibitors (NAIs), we measured their 50% inhibitory concentration (IC50) for influenza virus isolates from the 2014-2015 influenza season for comparison with those circulating in the 2010-2011 to 2013-2014 influenza seasons. Viral isolation was done with specimens obtained prior to treatment, and the type and subtype of influenza was determined by RT-PCR using type- and subtype-specific primers. The IC50 was determined by a neuraminidase inhibition assay using a fluorescent substrate. IC50 was measured for 200 influenza A(H3N2) and 19 influenza B in the 2014-2015 season, and no virus with highly reduced sensitivity to the four NAIs was detected. The ratios of the geometric means of the A(H3N2) IC50s of 2014-2015 to those of the 2010-2011, 2011-2012, 2012-2013, and 2013-2014 seasons ranged from 0.72 to 1.05, 0.82 to 1.22, 0.69 to 1.00, and 0.70 to 1.03, respectively. The ratios of the geometric mean of the B IC50s to the previous four seasons ranged from 0.59 to 1.28, 0.66 to 1.34, 0.84 to 1.21, and 1.06 to 1.47, respectively. There was no trend in the change of the IC50s for A(H3N2) or B. Significant differences were found in some seasons, but the differences in the IC50s were all less than two fold. These results show change in the geometric mean IC50 by season but with no trend, which indicates that the influence of viral mutation on the effectiveness of these NAIs was minute for A(H3N2) and B over the past five seasons.

  6. Development of 3-Phenyltropane Analogs with High Affinity for the Dopamine and Serotonin Transporters and Low Affinity for the Norepinephrine Transporter

    PubMed Central

    Jin, Chunyang; Navarro, Hernán A.; Carroll, F. Ivy

    2010-01-01

    Previous studies showed that the mixed monoamine transporter inhibitor (6, RTI-112) reduced cocaine self-administration at a high level of serotonin transporter (5-HTT) occupancy with no detectable dopamine transporter (DAT) occupancy. In this study, a series of 3β-(substituted phenyl)tropane-2β-carboxylic acid methyl esters 7a-g, 3β-(4-methoxyphenyl)tropane-2β-carboxylic acid esters 8a-j, and 3β-(4-methoxyphenyl)-2β-[3-(4′-methylphenyl)isoxazol-5-yl]tropane (9) were synthesized and evaluated for their monoamine transporter binding affinities to identify potent and selective compounds for both the DAT and 5-HTT relative to the norepinephrine transporter (NET). A number of compounds showed high binding affinities for both the DAT and 5-HTT and low affinity for the NET. 3β-(4-Methoxyphenyl)tropane-2β-carboxylic acid 2-(3-iodo-4-aminophenyl)ethyl ester (8i) with an IC50 value of 2.5 nM for the DAT and Ki values of 3.5 nM and 2040 nM for the 5-HTT and NET, respectively, is the most potent and selective compound for the DAT and 5-HTT relative to the NET in this study. PMID:19053748

  7. One-step purification of lactoperoxidase from bovine milk by affinity chromatography.

    PubMed

    Atasever, Ali; Ozdemir, Hasan; Gulcin, Ilhami; Irfan Kufrevioglu, O

    2013-01-15

    Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) of 0.848.10(-5)M. The K(i) for sulphanilamide was determined to be 3.57.10(-5)M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R(z) (A(412)/A(280)) value. The R(z) value for the purified LPO was found to be 0.7. Maximum binding was achieved and K(m) and V(max) values were determined.

  8. Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity.

    PubMed

    M'Barek, Sarrah; Chagot, Benjamin; Andreotti, Nicolas; Visan, Violeta; Mansuelle, Pascal; Grissmer, Stephan; Marrakchi, Mohamed; El Ayeb, Mohamed; Sampieri, François; Darbon, Hervé; Fajloun, Ziad; De Waard, Michel; Sabatier, Jean-Marc

    2005-08-15

    Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a "gain of function" approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well-characterized maurotoxin with its variant Pi1-like disulfide bridging (MTX(Pi1)), used as a molecular template, and butantoxin (BuTX), used as an N-terminal domain provider. BuTX is found to be 60-fold less potent than MTX(Pi1) in blocking Kv1.2 (IC(50) values of 165 nM for BuTX versus 2.8 nM for MTX(Pi1)). Removal of its N-terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6-fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX-Kv1.2 interaction. Transfer of the BuTX N-terminal domain to MTX(Pi1) results in a chimera with five disulfide bridges (BuTX-MTX(Pi1)) that exhibits 22-fold greater affinity for Kv1.2 than MTX(Pi1) itself, in spite of the lower affinity of BuTX as compared to MTX(Pi1). Docking experiments performed with the 3-D structure of BuTX-MTX(Pi1) in solution, as solved by (1)H-NMR, reveal that the N-terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity.

  9. The oxygen affinity of cytochrome bo' in Escherichia coli determined by the deoxygenation of oxyleghemoglobin and oxymyoglobin: Km values for oxygen are in the submicromolar range.

    PubMed Central

    D'Mello, R; Hill, S; Poole, R K

    1995-01-01

    Apparent oxygen affinities for Escherichia coli cells and membranes containing a terminal oxidase with only one type of ligand-binding heme, cytochrome o', were measured with oxyleghemoglobin and oxymyoglobin as sensitive oxygen reporters. Two Km values (0.15 to 0.35 microM and 0.016 to 0.085 microM) were detected, well below values determined for the purified oxidase by insensitive electrode methods. PMID:7836332

  10. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    PubMed Central

    Marcatili, Paolo; Pagnani, Andrea

    2016-01-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10−6), outperforming other sequence- and structure-based models. PMID:27074145

  11. Does cytotoxicity of metallointercalators correlate with cellular uptake or DNA affinity?

    PubMed

    Davis, Kimberley J; Carrall, Judith A; Lai, Barry; Aldrich-Wright, Janice R; Ralph, Stephen F; Dillon, Carolyn T

    2012-08-21

    The cytotoxicity of the metallointercalators, [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1R,2R-diaminocyclohexane)](2+) ([56MERR]) and [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1S,2S-diaminocyclohexane)](2+) ([56MESS]), towards A549 human lung cancer cells was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) value obtained following exposure of A549 cells to [56MESS] for 4 h was approximately three times smaller than that obtained when [56MERR] was administered under the same conditions, indicating that the former complex displayed greater cytotoxicity. Both IC(50) values were greater than that obtained after exposure of A549 cells to cisplatin, demonstrating that the latter compound was the most cytotoxic of the three examined. Microprobe synchrotron radiation X-ray fluorescence (SR-XRF) analyses of metallointercalator-treated A549 cells showed that platinum became localised in DNA-rich regions of the nucleus. In contrast, when the same cells were treated with cisplatin the metal became distributed throughout the cell. Determination of the maximum concentration of platinum present inside the cells using graphite furnace atomic absorption spectrophotometry (GFAAS) of platinum-treated cells suggested that there was greater uptake of [56MERR] compared to [56MESS] by the A549 cells, and that platinum uptake did not account for the greater toxicity of [56MESS], as assessed by the MTT assay. Electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic studies of solutions containing either [56MERR] or [56MESS], and a duplex hexadecamer molecule, showed the two metallointercalators displayed very similar affinity towards the nucleic acid. Overall these results indicate that the difference in cytotoxicity of the two platinum metallointercalators is probably the result of variations in their interactions with other cellular components. PMID:22740039

  12. Influence of length and flexibility of spacers on the binding affinity of divalent ligands.

    PubMed

    Liese, Susanne; Netz, Roland R

    2015-01-01

    We present a quantitative model for the binding of divalent ligand-receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.

  13. Influence of length and flexibility of spacers on the binding affinity of divalent ligands

    PubMed Central

    Liese, Susanne

    2015-01-01

    Summary We present a quantitative model for the binding of divalent ligand–receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket. PMID:26124882

  14. Purification of peroxidase from red cabbage (Brassica oleracea var. capitata f. rubra) by affinity chromatography.

    PubMed

    Somtürk, Burcu; Kalın, Ramazan; Özdemir, Nalan

    2014-08-01

    Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9% from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K m and V max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC50 and K i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702±0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.

  15. Proteochemometric modelling coupled to in silico target prediction: an integrated approach for the simultaneous prediction of polypharmacology and binding affinity/potency of small molecules.

    PubMed

    Paricharak, Shardul; Cortés-Ciriano, Isidro; IJzerman, Adriaan P; Malliavin, Thérèse E; Bender, Andreas

    2015-01-01

    The rampant increase of public bioactivity databases has fostered the development of computational chemogenomics methodologies to evaluate potential ligand-target interactions (polypharmacology) both in a qualitative and quantitative way. Bayesian target prediction algorithms predict the probability of an interaction between a compound and a panel of targets, thus assessing compound polypharmacology qualitatively, whereas structure-activity relationship techniques are able to provide quantitative bioactivity predictions. We propose an integrated drug discovery pipeline combining in silico target prediction and proteochemometric modelling (PCM) for the respective prediction of compound polypharmacology and potency/affinity. The proposed pipeline was evaluated on the retrospective discovery of Plasmodium falciparum DHFR inhibitors. The qualitative in silico target prediction model comprised 553,084 ligand-target associations (a total of 262,174 compounds), covering 3,481 protein targets and used protein domain annotations to extrapolate predictions across species. The prediction of bioactivities for plasmodial DHFR led to a recall value of 79% and a precision of 100%, where the latter high value arises from the structural similarity of plasmodial DHFR inhibitors and T. gondii DHFR inhibitors in the training set. Quantitative PCM models were then trained on a dataset comprising 20 eukaryotic, protozoan and bacterial DHFR sequences, and 1,505 distinct compounds (in total 3,099 data points). The most predictive PCM model exhibited R (2) 0 test and RMSEtest values of 0.79 and 0.59 pIC50 units respectively, which was shown to outperform models based exclusively on compound (R (2) 0 test/RMSEtest = 0.63/0.78) and target information (R (2) 0 test/RMSEtest = 0.09/1.22), as well as inductive transfer knowledge between targets, with respective R (2) 0 test and RMSEtest values of 0.76 and 0.63 pIC50 units. Finally, both methods were integrated to predict the protein

  16. A pharmacological analysis of high-affinity sodium transport in barley (Hordeum vulgare L.): a 24Na+/42K+ study

    PubMed Central

    Schulze, Lasse M.; Britto, Dev T.; Li, Mingyuan; Kronzucker, Herbert J.

    2012-01-01

    Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na+ uptake in this ‘high-affinity’ range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na+ and K+ fluxes were investigated using the short-lived radiotracers 24Na and 42K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na+ uptake was extremely sensitive to all treatments, displaying properties of high-affinity K+ transporters, K+ channels, animal Na+ channels, and non-selective cation channels. K+, NH4+NH4+, and Ca2+ suppressed Na+ transport biphasically, yielding IC50 values of 30, 10, and <5 μM, respectively. Reciprocal experiments showed that K+ influx is neither inhibited nor stimulated by Na+. Sodium efflux constituted 65% of influx, indicating a futile cycle. The thermodynamic feasibility of passive channel mediation is supported by compartmentation and electrophysiological data. Our study complements recent advances in the molecular biology of high-affinity Na+ transport by uncovering new physiological foundations for this transport phenomenon, while questioning its ecological relevance. PMID:22268152

  17. 3-Chlorotyramine Acting as Ligand of the D2 Dopamine Receptor. Molecular Modeling, Synthesis and D2 Receptor Affinity.

    PubMed

    Angelina, Emilio; Andujar, Sebastian; Moreno, Laura; Garibotto, Francisco; Párraga, Javier; Peruchena, Nelida; Cabedo, Nuria; Villecco, Margarita; Cortes, Diego; Enriz, Ricardo D

    2015-01-01

    We synthesized and tested 3-chlorotyramine as a ligand of the D2 dopamine receptor. This compound displayed a similar affinity by this receptor to that previously reported for dopamine. In order to understand further the experimental results we performed a molecular modeling study of 3-chlorotyramine and structurally related compounds. By combining molecular dynamics simulations with semiempirical (PM6), ab initio and density functional theory calculations, a simple and generally applicable procedure to evaluate the binding energies of these ligands interacting with the D2 dopamine receptors is reported here. These results provided a clear picture of the binding interactions of these compounds from both structural and energetic view points. A reduced model for the binding pocket was used. This approach allowed us to perform more accurate quantum mechanical calculations as well as to obtain a detailed electronic analysis using the Quantum Theory of Atoms in Molecules (QTAIM) technique. Molecular aspects of the binding interactions between ligands and the D2 dopamine receptor are discussed in detail. A good correlation between the relative binding energies obtained from theoretical calculations and experimental IC50 values was obtained. These results allowed us to predict that 3-chlorotyramine possesses a significant affinity by the D2 -DR. Our theoretical predictions were experimentally corroborated when we synthesized and tested 3-chlorotyramine which displayed a similar affinity by the D2 -DR to that reported for DA.

  18. A new tripodal receptor for molecular recognition of monosaccharides. A paradigm for assessing glycoside binding affinities and selectivities by 1H NMR spectroscopy.

    PubMed

    Vacca, Alberto; Nativi, Cristina; Cacciarini, Martina; Pergoli, Roberto; Roelens, Stefano

    2004-12-22

    A new tripodal receptor for the recognition of monosaccharides is described. The prototypical host 1 features a 1,3,5-substituted 2,4,6-triethylbenzene scaffold bearing three convergent H-bonding units. The binding ability of the t-octyl derivative 1a toward a set of octylglycosides of biologically relevant monosaccharides, including Glc, Gal, Man, and GlcNAc, was investigated by 1H NMR in CDCl3. A protocol for the correct evaluation of binding affinities was established, which can be generally applied for the recognition of monosaccharides by 1H NMR spectroscopy. A three-constant equilibrium model, including 1:1 and 2:1 host-guest association and dimerization of the receptor, was ascertained for the interaction of 1a with all the investigated glycosides. An affinity index, which we defined median binding concentration BC50 in analogy to the IC50 parameter, intended to address the general issue of comparing dimensionally heterogeneous binding data, and a limiting BC0(50)quantity describing intrinsic binding affinities were developed for evaluating the results. BC0(50) values for 1a range from 1 to 6 mM, indicating an intrinsic binding affinity in the millimolar range and a selectivity factor of 5 toward the investigated glycosides. The treatment has been extended to include any generic host-guest system involved in single or multiple binding equilibria.

  19. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  20. Binding affinity and agonist activity of putative endogenous cannabinoids at the human neocortical CB1 receptor.

    PubMed

    Steffens, Marc; Zentner, Josef; Honegger, Jürgen; Feuerstein, Thomas J

    2005-01-01

    We investigated the affinity of putative endocannabinoids (2-arachidonylglycerol, 2-AG; noladin ether, virodhamine) for the human neocortical CB1 receptor. Functional activity of these compounds (including anandamide, AEA) was determined by examining basal and forskolin-stimulated cAMP formation. Assays were performed with synaptosomes, prepared from fresh human neocortical tissue. Receptor affinity was assessed from competition binding experiments with the CB1/2 agonist [3H]-CP55.940 in absence or presence of a protease inhibitor to assess enzymatic stability. Noladin ether and virodhamine inhibited [3H]-CP55.940 binding (Ki: 98, 1740 nM, respectively). Protease inhibition decreased the Ki value of virodhamine (Ki: 912 nM), but left that of noladin ether unchanged. 2-AG almost lacked affinity (Ki lymphoblasic )10 microM). Basal cAMP formation was unaffected by AEA and noladin ether, but strongly enhanced by 2-AG and virodhamine. Forskolin-stimulated cAMP formation was inhibited by AEA and noladin ether (IC50: 69, 427 nM, respectively) to the same extent as by CP55.940 (Imax each approximately 30%). Inhibitions by AEA or noladin ether were blocked by the CB1 receptor antagonist AM251. Virodhamine increased forskolin-stimulated cAMP formation, also in presence of AM251, by approximately 20%. 2-AG had no effect; in presence of AM251, however, 10 microM 2-AG stimulated cAMP formation by approximately 15%. Our results suggest, that AEA and noladin ether are full CB1 receptor agonists in human neocortex, whereas virodhamine may act as a CB1 receptor antagonist/inverse agonist. Particularly the (patho)physiological role of 2-AG should be further investigated, since its CB1 receptor affinity and agonist activity especially in humans might be lower than generally assumed. PMID:15588725

  1. Bethe Ansatz and the Spectral Theory of Affine Lie Algebra-Valued Connections I. The simply-laced Case

    NASA Astrophysics Data System (ADS)

    Masoero, Davide; Raimondo, Andrea; Valeri, Daniele

    2016-06-01

    We study the ODE/IM correspondence for ODE associated to {widehat{mathfrak{g}}}-valued connections, for a simply-laced Lie algebra {mathfrak{g}}. We prove that subdominant solutions to the ODE defined in different fundamental representations satisfy a set of quadratic equations called {Ψ}-system. This allows us to show that the generalized spectral determinants satisfy the Bethe Ansatz equations.

  2. The adenosine receptor affinities and monoamine oxidase B inhibitory properties of sulfanylphthalimide analogues.

    PubMed

    Van der Walt, Mietha M; Terre'Blanche, Gisella; Petzer, Anél; Petzer, Jacobus P

    2015-04-01

    Based on a report that sulfanylphthalimides are highly potent monoamine oxidase (MAO) B selective inhibitors, the present study examines the adenosine receptor affinities and MAO-B inhibitory properties of a series of 4- and 5-sulfanylphthalimide analogues. Since adenosine antagonists (A1 and A2A subtypes) and MAO-B inhibitors are considered agents for the therapy of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs that antagonize adenosine receptors and inhibit MAO-B may have enhanced therapeutic value. The results document that the sulfanylphthalimide analogues are selective for the adenosine A1 receptor over the A2A receptor subtype, with a number of compounds also possessing MAO-B inhibitory properties. Among the compounds evaluated, 5-[(4-methoxybenzyl)sulfanyl]phthalimide was found to possess the highest binding affinity to adenosine A1 receptors with a Ki value of 0.369 μM. This compound is reported to also inhibit MAO-B with an IC50 value of 0.020 μM. Such dual-target-directed compounds may act synergistic in the treatment of Parkinson's disease: antagonism of the A1 receptor may facilitate dopamine release, while MAO-B inhibition may reduce dopamine metabolism. Additionally, dual-target-directed compounds may find therapeutic value in Alzheimer's disease: antagonism of the A1 receptor may be beneficial in the treatment of cognitive dysfunction, while MAO-B inhibition may exhibit neuroprotective properties. In neurological diseases, such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs are expected to be advantageous over single-target treatments.

  3. Time-Resolved Cell Culture Assay Analyser (TReCCA Analyser) for the Analysis of On-Line Data: Data Integration—Sensor Correction—Time-Resolved IC50 Determination

    PubMed Central

    Werner, Tobias; Wölfl, Stefan

    2015-01-01

    Time-resolved cell culture assays circumvent the need to set arbitrary end-points and reveal the dynamics of quality controlled experiments. However, they lead to the generation of large data sets, which can represent a complexity barrier to their use. We therefore developed the Time-Resolved Cell Culture Assay (TReCCA) Analyser program to perform standard cell assay analyses efficiently and make sophisticated in-depth analyses easily available. The functions of the program include data normalising and averaging, as well as smoothing and slope calculation, pin-pointing exact change time points. A time-resolved IC50/EC50 calculation provides a better understanding of drug toxicity over time and a more accurate drug to drug comparison. Finally the logarithmic sensor recalibration function, for sensors with an exponential calibration curve, homogenises the sensor output and enables the detection of low-scale changes. To illustrate the capabilities of the TReCCA Analyser, we performed on-line monitoring of dissolved oxygen in the culture media of the breast cancer cell line MCF-7 treated with different concentrations of the anti-cancer drug Cisplatin. The TReCCA Analyser is freely available at www.uni-heidelberg.de/fakultaeten/biowissenschaften/ipmb/biologie/woelfl/Research.html. By introducing the program, we hope to encourage more systematic use of time-resolved assays and lead researchers to fully exploit their data. PMID:26110644

  4. Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures.

    PubMed

    Wang, Haibo; Zhao, Xiaoping; Wang, Shufang; Tao, Shan; Ai, Ni; Wang, Yi

    2015-05-01

    Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase inhibitors have been used in the management of obesity. We present the first report on the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the selective enrichment of lipase inhibitors from natural products. A simple and rapid approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in 90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach were validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with high performance liquid chromatography-mass spectrometry technique to screen lipase inhibitors from the Magnoliae cortex extract, a medicinal plant used for treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 96.96 μM, respectively. The ligand-enzyme interactions of magnaldehyde B were further investigated by molecular docking. Our findings proved that enzyme-adsorbed nanotube could be used as a feasible and selective affinity medium for the rapid screening of enzyme inhibitors from complex mixtures.

  5. Evaluation of crocin and curcumin affinity on mushroom tyrosinase using surface plasmon resonance.

    PubMed

    Patil, Sushama; Srinivas, Sistla; Jadhav, Jyoti

    2014-04-01

    Tyrosinase inhibitors have potential applications in the cosmetics and food industries for preventing browning reactions and also as therapeutic drugs for neurodegenerative diseases such as Parkinson's. In this article, crocin and curcumin were evaluated as mushroom tyrosinase inhibitors. Results showed that, both compounds strongly inhibited the diphenolase activity than monophenolase. The IC50 values for diphenolase activity were estimated to be 0.11 mM and 0.18 mM for crocin and curcumin respectively. The binding kinetics of crocin and curcumin was studied with mushroom tyrosinase using surface plasmon resonance (SPR). Tyrosinase was immobilized on the gold surface of a Biacore sensor chip through amine coupling. Binding of inhibitors was analyzed by SPR without the need to further modify the surface or the use of other reagents. The binding constant KD (M) for mushroom tyrosinase obtained was 1.21×10(-4) M for crocin and 1.64×10(-4) M for curcumin, while showing a higher affinity for L-DOPA 1.95×10(-8) M, a substrate for tyrosinase (positive control). The study reveals the SPR sensor's ability to detect binding of the inhibitors.

  6. Evaluation of crocin and curcumin affinity on mushroom tyrosinase using surface plasmon resonance.

    PubMed

    Patil, Sushama; Srinivas, Sistla; Jadhav, Jyoti

    2014-04-01

    Tyrosinase inhibitors have potential applications in the cosmetics and food industries for preventing browning reactions and also as therapeutic drugs for neurodegenerative diseases such as Parkinson's. In this article, crocin and curcumin were evaluated as mushroom tyrosinase inhibitors. Results showed that, both compounds strongly inhibited the diphenolase activity than monophenolase. The IC50 values for diphenolase activity were estimated to be 0.11 mM and 0.18 mM for crocin and curcumin respectively. The binding kinetics of crocin and curcumin was studied with mushroom tyrosinase using surface plasmon resonance (SPR). Tyrosinase was immobilized on the gold surface of a Biacore sensor chip through amine coupling. Binding of inhibitors was analyzed by SPR without the need to further modify the surface or the use of other reagents. The binding constant KD (M) for mushroom tyrosinase obtained was 1.21×10(-4) M for crocin and 1.64×10(-4) M for curcumin, while showing a higher affinity for L-DOPA 1.95×10(-8) M, a substrate for tyrosinase (positive control). The study reveals the SPR sensor's ability to detect binding of the inhibitors. PMID:24444880

  7. Affinity and efficacy of racemic, (+)-, and (-)-methacholine in muscarinic inhibition of [3H]-noradrenaline release.

    PubMed

    Fuder, H; Jung, B

    1985-02-01

    The right postganglionic sympathetic nerves of rat isolated perfused hearts (previously loaded with [3H]-noradrenaline) were stimulated electrically with 10 trains of 10 pulses at 10 Hz. The inhibition by methacholine of stimulation-evoked [3H]-noradrenaline overflow into the perfusate (determined in the presence of corticosterone, desipramine, phentolamine, and propranolol) was taken as a measure for activation of presynaptic muscarinic receptors. The evoked [3H]-noradrenaline overflow was inhibited by (+)-, racemic, and (-)-methacholine in a reversible and concentration-dependent manner. The concentration causing 50% inhibition (IC50) was 0.1, 0.26, and 65 microM, respectively, resulting in an isomeric potency ratio IC50 (+)/IC50(-) of 650. The dissociation constant KA of the (+/-)- or (+)-methacholine-presynaptic receptor complex was determined after fractional receptor inactivation according to Furchgott & Bursztyn (1967) with phenoxybenzamine or propylbenzilylcholine mustard as irreversible antagonists of muscarinic receptors. KA for (-)-methacholine was estimated according to Mackay (1966). KA of (+)-, (+/-)-, and (-)-methacholine were 2.5, 4 and 440 microM, resulting in an isomeric affinity ratio KA (+)/KA(-) of 180. The discrepancy between the isomeric IC50 ratio and the isomeric KA ratio is explained by a higher intrinsic efficacy of the (+)-enantiomer compared to the (-)-enantiomer. Thus, (+)-methacholine has to occupy fewer receptors to induce a given inhibition of release than its antipode as revealed by a plot of fractional receptor occupancy vs response. The results show that, in the effector system of presynaptic muscarinic inhibition, methacholine enantiomers differ greatly not only in affinity for the receptor, but also to some extent in the efficiency of signal transmission, and both parameters contribute to the high isomeric potency ratio. The activity of the racemate is fully accounted for by the activity of the (+)-enantiomer.

  8. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    PubMed

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  9. Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor.

    PubMed

    Liu, Yen-Chin; Liu, Yi-Liang; Su, Jia-Yang; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities. PMID:21552531

  10. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells

    PubMed Central

    Bhatia, Prateek A.; Moaddel, Ruin; Wainer, Irving W.

    2010-01-01

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodopetra frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp c-DNA, using a baculovirus expression system. The resulting CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [3H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9MRP1) column, etoposide and furosemide on the CMAC(Sf9MRP2) column and etoposide and fumitremorgin C on the CMAC(Sf9BCPR) column The binding affinities (Ki values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [3H]-etoposide on the CMAC(Sf9MRP1) column to a greater extent than (R)-verapamil and the relative IC50 values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC50 values were consistent with previously reported data. The results indicated that the CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system. PMID:20441926

  11. Development of an AlphaScreen assay for discovery of inhibitors of low-affinity glycan-lectin interactions.

    PubMed

    Yegorova, Svetlana; Chavaroche, Anais E; Rodriguez, Maria C; Minond, Dmitriy; Cudic, Mare

    2013-08-15

    The development of high-throughput screening (HTS) assays with increased sensitivity for the identification of potent and selective inhibitors of galectins has been hampered by the weak binding affinities between galectins and their carbohydrate ligands. To circumvent this obstacle, we have developed an AlphaScreen assay for a 384-well plate format in a competitive binding configuration for discovery of new inhibitors of galectin-3. His-tagged galectin-3 was bound to nickel chelate acceptor beads, whereas biotinylated asialofetuin (biotin-ASF), a galectin-3 nanomolar binding partner, was bound to streptavidin-coated donor beads. Inhibitors of the carbohydrate-galectin interaction lead to a reduction of the AlphaScreen signal by competing with the biotin-ASF. The obtained IC50 values for known carbohydrate ligands of galectin-3 are in good agreement with the Kd values reported and measured for galectin-3 by isothermal titration calorimetry (ITC). Thus, the developed AlphaScreen assay in a competitive binding configuration offers several advantages over the existing screening assays for inhibitors of glycan-lectin interactions. In addition, the assay format for the galectin-3/ASF pair could be easily applied in screening for glycan- and/or small molecule-based inhibitors of other members of the galectin family.

  12. Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo

    PubMed Central

    Botkjaer, Kenneth A.; Kwok, Hang Fai; Terp, Mikkel G.; Karatt-Vellatt, Aneesh; Santamaria, Salvatore; McCafferty, John; Andreasen, Peter A.; Itoh, Yoshifumi; Ditzel, Henrik J.; Murphy, Gillian

    2016-01-01

    The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332-fold with the ability to interfere with cell-surface MT1-MMP functions, displaying IC50 values down to 5 nM. Importantly, the new inhibitors were able to inhibit collagen invasion by tumor-cells in vitro and in vivo primary tumor growth and metastasis of MDA-MB-231 cells in a mouse orthotopic xenograft model. Herein is the first demonstration that an inhibitory antibody targeting sites outside the catalytic cleft of MT1-MMP can effectively abrogate its in vivo activity during tumorigenesis and metastasis. PMID:26934448

  13. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11.

    PubMed

    Lundegaard, Claus; Lamberth, Kasper; Harndahl, Mikkel; Buus, Søren; Lund, Ole; Nielsen, Morten

    2008-07-01

    NetMHC-3.0 is trained on a large number of quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from SYFPEITHI. The method generates high-accuracy predictions of major histocompatibility complex (MHC): peptide binding. The predictions are based on artificial neural networks trained on data from 55 MHC alleles (43 Human and 12 non-human), and position-specific scoring matrices (PSSMs) for additional 67 HLA alleles. As only the MHC class I prediction server is available, predictions are possible for peptides of length 8-11 for all 122 alleles. artificial neural network predictions are given as actual IC(50) values whereas PSSM predictions are given as a log-odds likelihood scores. The output is optionally available as download for easy post-processing. The training method underlying the server is the best available, and has been used to predict possible MHC-binding peptides in a series of pathogen viral proteomes including SARS, Influenza and HIV, resulting in an average of 75-80% confirmed MHC binders. Here, the performance is further validated and benchmarked using a large set of newly published affinity data, non-redundant to the training set. The server is free of use and available at: http://www.cbs.dtu.dk/services/NetMHC.

  14. Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)phthalazine derivatives.

    PubMed

    Eldehna, Wagdy M; Abou-Seri, Sahar M; El Kerdawy, Ahmed M; Ayyad, Rezk R; Hamdy, Abdallah M; Ghabbour, Hazem A; Ali, Mamdouh M; Abou El Ella, Dalal A

    2016-05-01

    A series of anilinophthalazine derivatives 4a-j was initially synthesized and tested for its VEGFR-2 inhibitory activity where it showed promising activity (IC50 = 0.636-5.76 μM). Molecular docking studies guidance was used to improve the binding affinity for series 4a-j towards VEGFR-2 active site. This improvement was achieved by increasing the hydrophobic interaction with the hydrophobic back pocket of the VEGFR-2 active site lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044. Increasing the hydrophobic interaction was accomplished by extending the anilinophthalazine scaffold with a substituted phenyl moiety through an uriedo linker which should give this extension the flexibility required to accommodate itself deeply into the hydrophobic back pocket. As planned, the designed uriedo-anilinophthalazines 7a-i showed superior binding affinity than their anilinophthalazine parents (IC50 = 0.083-0.473 μM). In particular, compounds 7g-i showed IC50 of 0.086, 0.083 and 0.086 μM, respectively, which are better than that of the reference drug sorafenib (IC50 = 0.09 μM).

  15. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  16. Synthesis and structure-activity relationship studies of novel 2-diarylethyl substituted (2-carboxycycloprop-1-yl)glycines as high-affinity group II metabotropic glutamate receptor ligands.

    PubMed

    Sørensen, Ulrik S; Bleisch, Thomas J; Kingston, Anne E; Wright, Rebecca A; Johnson, Bryan G; Schoepp, Darryle D; Ornstein, Paul L

    2003-01-17

    The major excitatory neurotransmitter in the central nervous system, (S)-glutamic acid , activates both ionotropic and metabotropic excitatory amino acid receptors. Its importance in connection to neurological and psychiatric disorders has directed great attention to the development of compounds that modulate the effects of this endogenous ligand. Whereas L-carboxycyclopropylglycine (L-CCG-1) is a potent agonist at, primarily, group II metabotropic glutamate receptors, alkylation of at the alpha-carbon notoriously result in group II mGluR antagonists, of which the most potent compound described so far, LY341495, displays IC(50) values of 23 and 10 nM at the group II receptor subtypes mGlu2 and mGlu3, respectively. In this study we synthesized a series of structural analogues of in which the xanthyl moiety is replaced by two substituted-phenyl groups. The pharmacological characterization shows that these novel compounds have very high affinity for group II mGluRs when tested as their racemates. The most potent analogues demonstrate K(i) values in the range of 5-12 nM, being thus comparable to LY341495. PMID:12470714

  17. Inhibitory effects of hesperetin derivatives on guinea pig phosphodiesterases and their ratios between high- and low-affinity rolipram binding.

    PubMed

    Hsu, Hsin-Te; Wang, Wen-Hung; Han, Cheng-Ying; Chen, Chun-Nan; Chen, Chi-Ming; Ko, Wun-Chang

    2013-07-01

    The phosphodiesterase (PDE)4 molecule exists as two distinct conformers, PDE4H and PDE4L , which have high and low affinities, respectively, for the selective PDE4 inhibitor, rolipram. The inhibition of PDE4H and PDE4L is associated with adverse responses, such as nausea, vomiting, and gastric hypersecretion, and with anti-inflammatory and bronchodilator effects, respectively. We determined the therapeutic (PDE4H/PDE4L) ratios of hesperetin-7-O-methylether, hesperetin-5,7,3'-O-trimethylether (HTME), hesperetin-7-O-acetate, hesperetin-7,3'-O-diacetate, hesperetin-5,7,3'-O-triacetate (HTA), hesperetin-5,7,3'-O-tripropionate, hesperetin-5,7,3'-O-tributyrate, hesperetin-5,7,3'-O-triisobutyrate, and hesperetin-5,7,3'-O-tripivatate, and compared these ratios to those of hesperetin, hesperetin-7,3'-O-dimethylether, hesperidin, and hesperidin-3'-O-methylether to identify derivatives with therapeutic ratios and to characterize the structure-activity relationships among these compounds. The activities of PDE isozymes 1 through 5 were measured using a two-step procedure using [(3)H]adenosine 3',5'-cyclic monophosphate or [(3)H]guanosine 3',5'-cyclic monophosphate as substrates. The inhibitory concentration (IC50) for 50% of PDE4 inhibition and effective concentration (EC50) for replacing 50% of [(3)H]rolipram binding on high-affinity rolipram-binding sites was taken as the PDE4L and PDE4H value, respectively. The HTME and the HTA dually inhibited PDE3 and PDE4, and displayed PDE4H/PDE4L ratios of 18.3 and 20.8, respectively, suggesting that they may be candidate drugs for treating asthma and chronic obstructive pulmonary disease (COPD) because the combined inhibition of PDE3 and PDE4 has synergistically anti-inflammatory and bronchodilator effects in COPD patients.

  18. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

    PubMed Central

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-01-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  19. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-15

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

  20. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  1. Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, María del Mar; Alaiz, Manuel; Girón-Calle, Julio; Millan, Francisco; Vioque, Javier

    2006-09-20

    A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.

  2. High affinity P2x-purinoceptor binding sites for [35S]-adenosine 5'-O-[3-thiotriphosphate] in rat vas deferens membranes.

    PubMed Central

    Michel, A. D.; Humphrey, P. P.

    1996-01-01

    1. The binding sites labelled by [35S]-adenosine 5'-O-[3-thiotriphosphate]([35S]-ATP gamma S) at 4 degrees C in rat vas deferens membranes were studied and compared to the sites labelled by [3H]-alpha,beta-methylene ATP ([3H]-alpha beta meATP) to ascertain whether [35S]-ATP gamma S can be used to label the P2x purinoceptor. 2. In the presence of 4 mM CaCl2, the binding of 0.2 nM [35S]-ATP gamma S to vas deferens membranes was increased 3.4 fold, when compared to studies performed in the absence of calcium. However, binding did not appear to be solely to P2x purinoceptors since [35S]-ATP gamma S labelled a heterogeneous population of sites and about 72% of the sites possessed high affinity (pIC50 = 7.5) for guanosine 5'-O-[3-thiotriphosphate] (GTP gamma S). Even in the presence of 1 microM GTP gamma S, to occlude the sites with high affinity for GTP gamma S, the binding of [35S]-ATP gamma S was heterogeneous and since there was also evidence of extensive metabolism of ATP in the presence of calcium, the binding of [35S]-ATP gamma S under these conditions was not studied further. 3. In the absence of calcium ions, [35S]-ATP gamma S bound to a single population of sites (pKD = 9.23; Bmax = 4270 fmol mg-1 protein). Binding reached steady state within 3 h (t1/2 = 38 min), was stable for a further 4 h and was readily reversible upon addition of 10 microM unlabelled ATP gamma S (t1/2 = 45 min). In competition studies the binding of 0.2 nM [35S]-ATP gamma S was inhibited by a number of P2x purinoceptor agonists and antagonists, but not by adenosine receptor agonists, staurosporine (1 microM) or several ATPase inhibitors. The rank order of agonist affinity estimates (pIC50 values) in competing for the [35S]-ATP gamma S binding sites was: ATP (9.01), 2-methylthio- ATP (8.79), ATP gamma S (8.73), alpha beta meATP (7.57), ADP (7.24), beta, gamma-methylene ATP (7.18), L-beta, gamma-methylene ATP (5.83), alpha, beta-methylene ADP (4.36). 4. Affinity estimates (pIC50 values) for

  3. [3H]-RS-45041-190: a selective high-affinity radioligand for I2 imidazoline receptors.

    PubMed Central

    MacKinnon, A. C.; Redfern, W. S.; Brown, C. M.

    1995-01-01

    1. RS-45041-190 (4-chloro-2-(imidazolin-2-yl)isoindoline) is an I2 imidazoline receptor ligand with the highest affinity and selectivity so far described; [3H]-RS-45041-190 has a tritium atom attached to the 7-position on the isoindoline ring. 2. [3H]-RS-45041-190 binding to rat kidney membranes was saturable (Bmax = 223.1 +/- 18.4 fmol mg-1 protein) and of high affinity (Kd = 2.71 +/- 0.59 nM). Kinetic studies revealed that the binding was rapid and reversible, with [3H]-RS-45041-190 interacting with two sites or two affinity states. 3. Competition studies showed that 60-70% of [3H]-RS-45041-190 binding (1 nM) was specifically to imidazoline binding sites of the I2 subtype, characterized by high affinity for idazoxan (pIC50 7.85 +/- 0.03) and cirazoline (pIC50 8.16 +/- 0.05). The remaining 30-40% was displaced specifically by the monoamine oxidase A inhibitors, clorgyline and pargyline. 4. alpha 1- and alpha 2-adrenoceptor, I1 imidazoline, histamine, 5-hydroxytryptamine or dopamine receptor ligands had low affinity suggesting that [3H]-RS-45041-190 did not label receptors of these classes. 5. In autoradiography studies, [3H]-RS-45041-190 labelled discrete regions of rat brain corresponding to the distribution of I2 subtypes, notably the subfornical organ, arcuate nucleus, interpeduncular nucleus, medial habenular nucleus and lateral mammillary nucleus, and additional sites in the locus coeruleus, dorsal raphe and dorsomedial hypothalamic nucleus. 6. [3H]-RS-45041-190 therefore labels I2 receptors with high affinity, and an additional site which has high affinity for some monoamine oxidase inhibitors. Images Figure 6 Figure 7 Figure 8 PMID:8528552

  4. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  5. Characterization of the binding of a novel nonxanthine adenosine antagonist radioligand, ( sup 3 H)CGS 15943, to multiple affinity states of the adenosine A1 receptor in the rat cortex

    SciTech Connect

    Jarvis, M.F.; Williams, M.; Do, U.H.; Sills, M.A. )

    1991-01-01

    The triazoloquinazoline CGS 15943 is the first reported nonxanthine adenosine antagonist that has high affinity for brain adenosine receptors. In the present study, the binding of (3H) CGS 15943 to recognition sites in rat cortical membranes was characterized. Saturation experiments revealed that (3H)CGS 15943 labeled a single class of recognition sites with high affinity and limited capacity. Competition studies revealed that the binding of (3H)CGS 15943 was consistent with the labeling of brain adenosine A1 receptors. Adenosine agonists inhibited 1 nM (3H)CGS 15943 binding with the following order of activity N6-cyclopentyladenosine (IC50 = 15 nM) greater than 2-chloroadenosine greater than (R)-N6-phenylisopropyladenosine greater than 5'-N6-ethylcarboxamidoadenosine greater than (S)N6-phenylisopropyladenosine greater than CGS 21680 greater than CV 1808 (IC50 greater than 10,000 nM). The potency order for adenosine antagonists was CGS 15943 (IC50 = 5 nM) greater than 8-phenyltheophylline greater than 1,3-dipropyl-8-(4-amino-2-chloro)phenylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than theophylline = caffeine (IC50 greater than 10,000 nM). Antagonist inhibition curves were steep and best described by a one-site binding model. In contrast, adenosine A1 agonist competition curves were shallow, as indicated by Hill coefficients less than unity. Computer analysis revealed that these inhibition curves were best described by a two-site binding model. Agonist competition curves generated in the presence of 1 mM GTP resulted in a rightward shift and steepening of the inhibition-concentration curves, whereas antagonist binding was not altered in the presence of GTP. The complex binding interactions found with adenosine agonists indicate that (3H)CGS 15943 labels both high and low affinity components of the adenosine A1 receptor in the rat cortex.

  6. Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

    PubMed Central

    Rosa, Sofia Tabares-da; Rossotti, Martin; Carleiza, Carmen; Carrión, Federico; Pritsch, Otto; Ahn, Ki Chang; Last, Jerold A.; Hammock, Bruce D; González-Sapienza, Gualberto

    2011-01-01

    Single-domain antibodies (sdAbs) found in camelids, lack a light chain and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. While this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC50 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, KD 0.98–1.37 nM (SPR) which allowed development of a competitive assay for TCC with an IC50 = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC50 attained with other anti-hapten VHHs reported thus far. Despite the modest overall anti-hapten sdAbs response in llamas, a small subpopulation of high affinity VHHs are generated that can be isolated by carefully design of the selection process. PMID:21827167

  7. Characterization of the affinities of insulin-like growth factor (IGF)-binding proteins 1-4 for IGF-I, IGF-II, IGF-I/insulin hybrid, and IGF-I analogs.

    PubMed

    Oh, Y; Müller, H L; Lee, D Y; Fielder, P J; Rosenfeld, R G

    1993-03-01

    Insulin-like growth factor (IGF)-binding proteins (BPs) bind IGF-I and IGF-II with high affinity and modify the activity of IGF peptides in a complex manner. We have characterized the affinities of IGFBP-1-4 for IGF-I and -II by employing 1) purified IGFBP preparations, 2) both [125I]IGF-I and [125I]IGF-II as radioligands, and 3) multiple IGF analogs designed to have altered affinities for IGFBPs. To this end, human (h) IGFBP-1, hIGFBP-2, and rat (r) IGFBP-4 have been purified to homogeneity from human amniotic fluid, human prostate epithelial cell culture, and B104 rat neuroblastoma cells; for human IGFBP-3, the glycosylated recombinant form (rec-hIGFBP-3), produced in Chinese hamster ovary cells, was employed. The IC50 values of IGF-I for hIGFBP-1, hIGFBP-2, rec-hIGFBP-3, rIGFBP-4, and human serum IGFBPs were 0.05 +/- 0.01, 5.0 +/- 0.01, 0.25 +/- 0.20, 0.6 +/- 0.4, and 0.1 +/- 0.01 ng/ml, respectively. While hIGFBP-1 and rIGFBP-4 had virtually equivalent affinities for IGF-I and IGF-II, hIGFBP-2 and rec-hIGFBP-3 demonstrated 2- to 5-fold higher affinities for IGF-II than for IGF-I. Studies with [Gln3,Ala4,Tyr15,Leu16]IGF-I and Des-(1-3)-IGF-I indicate that specific residues in the first 16 amino acids of the B domain of IGF-I appear to be critical for binding to all of the IGFBPs tested, but not to IGF receptors. However, severe modifications in the B domain disrupt binding affinity, not only for IGFBPs, but also for receptors (IGF-I/insulin hybrid and B-chain mutant). Interestingly, modifications in the A domain of IGF-I, which is believed to contain residues critical for binding to IGF-I and insulin receptors, show differential effects on binding affinity to BPs. [Thr49,Ser50,Ile51]IGF-I, which has normal affinity for the type I IGF receptor, shows at least a 500-fold decreased affinity for hIGFBP-1 and recombinant hIGFBP-3, in contrast to 50- to 100-fold reduced affinity for hIGFBP-2 and rIGFBP-4, and 5- to 10-fold reduced affinity for purified human serum

  8. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  9. Affine projective Osserman structures

    NASA Astrophysics Data System (ADS)

    Gilkey, P.; Nikčević, S.

    2013-08-01

    By considering the projectivized spectrum of the Jacobi operator, we introduce the concept of projective Osserman manifold in both the affine and in the pseudo-Riemannian settings. If M is an affine projective Osserman manifold, then the deformed Riemannian extension metric on the cotangent bundle is both spacelike and timelike projective Osserman. Since any rank-1-symmetric space is affine projective Osserman, this provides additional information concerning the cotangent bundle of a rank-1 Riemannian symmetric space with the deformed Riemannian extension metric. We construct other examples of affine projective Osserman manifolds where the Ricci tensor is not symmetric and thus the connection in question is not the Levi-Civita connection of any metric. If the dimension is odd, we use methods of algebraic topology to show the Jacobi operator of an affine projective Osserman manifold has only one non-zero eigenvalue and that eigenvalue is real.

  10. Omega-conotoxin GVIA binding to a high-affinity receptor in brain: characterization, calcium sensitivity, and solubilization

    SciTech Connect

    Wagner, J.A.; Snowman, A.M.; Biswas, A.; Olivera, B.M.; Snyder, S.H.

    1988-09-01

    We describe unique, high-affinity binding sites for omega(/sup 125/I)conotoxin GVIA in membranes from rat brain and rabbit sympathetic ganglia which appear to be primarily associated with N-type voltage-dependent calcium channels. The dissociation constant (KD) for the toxin in rat brain membranes is 60 pM. Physiologic extracellular concentrations of calcium inhibit toxin binding noncompetitively (IC50 = 0.2 mM). The regional distribution of the binding sites in rat brain differs markedly from that of dihydropyridine calcium antagonist receptors associated with L-type calcium channels. In detergent-solubilized brain membranes, toxin binding retains the same affinity, specificity, and ionic sensitivity as in particulate preparations.

  11. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  12. Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

    PubMed Central

    Marszałł, Michał P.; Moaddel, Ruin; Jozwiak, Krzysztof; Bernier, Michel; Wainer, Irving W.

    2008-01-01

    Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N-terminus to create the Hsp90α(NT)-column or C-terminus to create the Hsp90α(CT)-column. Binding to the exposed C-terminus on the Hsp90α(NT)-column was characterized using frontal chromatography and C-terminus ligands coumermycin A1(CA1) and novobiocin (NOVO). The calculated Kd values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Non-linear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex, 22.2 (±8.8) μM−1 sec−1 and 2.7 (±0.6) sec−1, respectively. Binding to the exposed N-terminus on the Hsp90α(CT)-column was characterized using frontal chromatography. The Kd values of N-terminus ligands geldanamycin (GM) (90 ± 50 nM) 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) (210 ± 50 nM) and radicicol (RAD) (20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC50 values for inhibition of ATPase activity on the Hsp90α(CT)-column. The IC50 for GM was 2.80 ± 0.18 μM and the relative IC50 values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition. PMID:18047824

  13. Identification of selective, high affinity [125I]-angiotensin and [125I]-bradykinin binding sites in rat intestinal epithelia.

    PubMed Central

    Cox, H. M.; Munday, K. A.; Poat, J. A.

    1986-01-01

    Specific [125I]-angiotensin II (AII) and [125I]-bradykinin (Bk) binding sites have been identified within epithelial membranes from rat jejunum and descending colon. These high affinity intestinal sites exhibited KD values of 0.64 +/- 0.16 nM for [125I]-AII and 0.69 +/- 0.13 nM for [125I]-Bk, which were similar to those for [125I]-AII (0.85 nM) and [125I]-Bk binding sites (1.03 nM) previously identified in renal cortex epithelia. Specific [125I]-AII binding capacity was only 19.77 +/- 2.74 fmol mg-1 in small intestine and 11.31 +/- 2.66 fmol mg-1 in descending colon epithelia while a larger population, 332.0 +/- 72.9 fmol-mg-1 of specific [125I]-Bk sites were identified in epithelial membranes from small intestine. Significant hydrolysis of both free [125I]-AII and [125I]-Bk was observed while membrane bound peptides remained relatively resistant to degradation. Whilst no corrections have been made to the observed values of KD and Bmax quoted above, one may assume that the calculated reductions in the free hormone concentration will result in a decrease of the KD value for both peptides. Loss of membrane bound peptide, particularly of [125I]-AII, may indicate that the calculated Bmax value is an underestimation. Despite the rapid degradation of unbound [125I]-AII and [125I]-Bk during incubations the kinetics of specific peptide binding were reversible and highly selective. The order of potency for specific [125I]-AII binding was [Sar1, Leu8]-AII greater than or equal to [Sar1, Thr8]-AII greater than or equal to AII greater than [Sar1, Ile8]-AII greater than or equal to [Des, Asp1, Ile8] AII greater than AIII. Specific [125I]-Bk binding was also highly selective, the order of potency being Phe8-Bk greater than or equal to Tyr8-Bk greater than or equal to Lys-Bk much greater than Des, Arg1-Bk. AII exhibited an IC50 of greater than 1mM for specific [125I]-Bk binding and likewise Phe8-Bk for specific [125I]-AII binding. PMID:2869810

  14. Affinity driven social networks

    NASA Astrophysics Data System (ADS)

    Ruyú, B.; Kuperman, M. N.

    2007-04-01

    In this work we present a model for evolving networks, where the driven force is related to the social affinity between individuals of a population. In the model, a set of individuals initially arranged on a regular ordered network and thus linked with their closest neighbors are allowed to rearrange their connections according to a dynamics closely related to that of the stable marriage problem. We show that the behavior of some topological properties of the resulting networks follows a non trivial pattern.

  15. Biological effect of varying peptide binding affinity to the BoLA-DRB3*2703 allele

    PubMed Central

    2003-01-01

    MHC class I and II molecules are immunoregulatory cell surface glycoproteins, which selectively bind to and present antigenic peptides to T-lymphocytes. Murine and human studies show that variable peptide binding affinity to MHC II molecules influences Th1/Th2 responses by inducing distinctive cytokine expression. To examine the biological effects of peptide binding affinity to bovine MHC (BoLA), various self peptides (BoLA-DQ and fibrinogen fragments) and non-self peptides from ovalbumin (OVA), as well as VP2 and VP4 peptides from foot and mouth disease virus (FMD-V) were used to (1) determine binding affinities to the BoLA-DRB3*2703 allele, previously associated with mastitis susceptibility and (2) determine whether peptide binding affinity influences T-lymphocyte function. Peptide binding affinity was determined by a competitive assay using high affinity biotinylated self-peptide incubated with purified BoLA-DRB3*2703 in the presence of various concentrations of competing peptides. The concentrations of non-self peptide required to inhibit self-peptide binding by 50% (IC50) were variable, ranging from 26.92 to > 320 μM. Peptide-specific T-lymphocyte function was determined by measuring DNA synthesis, cell division, and IFN-γ production in cultures of mononuclear cells from a BoLA-DRB3*2703 homozygous cow. When compared to non-stimulated control cultures, differences in lymphocyte function were observed for all of the assessed parameters; however, peptide-binding affinity did not always account for the observed differences in lymphocyte function. PMID:12927080

  16. Proton affinities of hydrated molecules

    NASA Astrophysics Data System (ADS)

    Valadbeigi, Younes

    2016-09-01

    Proton affinities (PA) of non-hydrated, M, and hydrated forms, M(H2O)1,2,3, of 20 organic molecules including alcohols, ethers, aldehydes, ketones and amines were calculated by the B3LYP/6-311++G(d,p) method. For homogeneous families, linear correlations were observed between PAs of the M(H2O)1,2,3 and the PAs of the non-hydrated molecules. Also, the absolute values of the hydration enthalpies of the protonated molecules decreased linearly with the PAs. The correlation functions predicted that for an amine with PA < 1100 kJ/mol the PA(M(H2O)) is larger than the corresponding PA, while for an amine with PA > 1100 kJ/mol the PA(M(H2O)) is smaller than the PA.

  17. Binding Kinetics versus Affinities in BRD4 Inhibition.

    PubMed

    Kuang, Ming; Zhou, Jingwei; Wang, Laiyou; Liu, Zhihong; Guo, Jiao; Wu, Ruibo

    2015-09-28

    Bromodomains (BRDs) are protein modules that selectively recognize histones as a "reader" by binding to an acetylated lysine substrate. The human BRD4 has emerged as a promising drug target for a number of disease pathways, and several potent BRD inhibitors have been discovered experimentally recently. However, the detailed inhibition mechanism especially for the inhibitor binding kinetics is not clear. Herein, by employing classical molecular dynamics (MD) and state-of-the-art density functional QM/MM MD simulations, the dynamic characteristics of ZA-loop in BRD4 are revealed. And then the correlation between binding pocket size and ZA-loop motion is elucidated. Moreover, our simulations found that the compound (-)-JQ1 could be accommodated reasonably in thermodynamics whereas it is infeasible in binding kinetics against BRD4. Its racemate (+)-JQ1 proved to be both thermodynamically reasonable and kinetically achievable against BRD4, which could explain the previous experimental results that (+)-JQ1 shows a high inhibitory effect toward BRD4 (IC50 is 77 nM) while (-)-JQ1 is inactive (>10 μM). Furthermore, the L92/L94/Y97 in the ZA-loop and Asn140 in the BC-loop are identified to be critical residues in (+)-JQ1 binding/releasing kinetics. All these findings shed light on further selective inhibitor design toward BRD family, by exploiting the non-negligible ligand binding kinetics features and flexible ZA-loop motions of BRD, instead of only the static ligand-protein binding affinity. PMID:26263125

  18. Binding Kinetics versus Affinities in BRD4 Inhibition.

    PubMed

    Kuang, Ming; Zhou, Jingwei; Wang, Laiyou; Liu, Zhihong; Guo, Jiao; Wu, Ruibo

    2015-09-28

    Bromodomains (BRDs) are protein modules that selectively recognize histones as a "reader" by binding to an acetylated lysine substrate. The human BRD4 has emerged as a promising drug target for a number of disease pathways, and several potent BRD inhibitors have been discovered experimentally recently. However, the detailed inhibition mechanism especially for the inhibitor binding kinetics is not clear. Herein, by employing classical molecular dynamics (MD) and state-of-the-art density functional QM/MM MD simulations, the dynamic characteristics of ZA-loop in BRD4 are revealed. And then the correlation between binding pocket size and ZA-loop motion is elucidated. Moreover, our simulations found that the compound (-)-JQ1 could be accommodated reasonably in thermodynamics whereas it is infeasible in binding kinetics against BRD4. Its racemate (+)-JQ1 proved to be both thermodynamically reasonable and kinetically achievable against BRD4, which could explain the previous experimental results that (+)-JQ1 shows a high inhibitory effect toward BRD4 (IC50 is 77 nM) while (-)-JQ1 is inactive (>10 μM). Furthermore, the L92/L94/Y97 in the ZA-loop and Asn140 in the BC-loop are identified to be critical residues in (+)-JQ1 binding/releasing kinetics. All these findings shed light on further selective inhibitor design toward BRD family, by exploiting the non-negligible ligand binding kinetics features and flexible ZA-loop motions of BRD, instead of only the static ligand-protein binding affinity.

  19. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  20. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  1. Compressed images for affinity prediction-2 (CIFAP-2): an improved machine learning methodology on protein-ligand interactions based on a study on caspase 3 inhibitors.

    PubMed

    Erdas, Ozlem; Andac, Cenk A; Gurkan-Alp, A Selen; Alpaslan, Ferda Nur; Buyukbingol, Erdem

    2015-01-01

    The aim of this study is to propose an improved computational methodology, which is called Compressed Images for Affinity Prediction-2 (CIFAP-2) to predict binding affinities of structurally related protein-ligand complexes. CIFAP-2 method is established based on a protein-ligand model from which computational affinity information is obtained by utilizing 2D electrostatic potential images determined for the binding site of protein-ligand complexes. The quality of the prediction of the CIFAP-2 algorithm was tested using partial least squares regression (PLSR) as well as support vector regression (SVR) and adaptive neuro-fuzzy ınference system (ANFIS), which are highly promising prediction methods in drug design. CIFAP-2 was applied on a protein-ligand complex system involving Caspase 3 (CASP3) and its 35 inhibitors possessing a common isatin sulfonamide pharmacophore. As a result, PLSR affinity prediction for the CASP3-ligand complexes gave rise to the most consistent information with reported empirical binding affinities (pIC(50)) of the CASP3 inhibitors. PMID:25578823

  2. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  3. Affinity based information diffusion model in social networks

    NASA Astrophysics Data System (ADS)

    Liu, Hongli; Xie, Yun; Hu, Haibo; Chen, Zhigao

    2014-12-01

    There is a widespread intuitive sense that people prefer participating in spreading the information in which they are interested. The affinity of people with information disseminated can affect the information propagation in social networks. In this paper, we propose an information diffusion model incorporating the mechanism of affinity of people with information which considers the fitness of affinity values of people with affinity threshold of the information. We find that the final size of information diffusion is affected by affinity threshold of the information, average degree of the network and the probability of people's losing their interest in the information. We also explore the effects of other factors on information spreading by numerical simulations and find that the probabilities of people's questioning and confirming the information can affect the propagation speed, but not the final scope.

  4. The sodium ion affinities of asparagine, glutamine, histidine and arginine

    NASA Astrophysics Data System (ADS)

    Wang, Ping; Ohanessian, Gilles; Wesdemiotis, Chrys

    2008-01-01

    The sodium ion affinities of the amino acids Asn, Gln, His and Arg have been determined by experimental and computational approaches (for Asn, His and Arg). Na+-bound heterodimers with amino acid and peptide ligands (Pep1, Pep2) were produced by electrospray ionization. From the dissociation kinetics of these Pep1-Na+-Pep2 ions to Pep1-Na+ and Pep2-Na+, determined by collisionally activated dissociation, a ladder of relative affinities was constructed and subsequently converted to absolute affinities by anchoring the relative values to known Na+ affinities. The Na+ affinities of Asn, His and Arg, were calculated at the MP2(full)/6-311+G(2d,2p)//MP2/6-31G(d) level of ab initio theory. The resulting experimental and computed Na+ affinities are in excellent agreement with one another. These results, combined with those of our previous studies, yield the sodium ion affinities of 18 out of the 20 [alpha]-amino acids naturally occurring in peptides and proteins of living systems.

  5. Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

    SciTech Connect

    Borst, S.E.; Catherwood, B.D. )

    1989-04-01

    We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of (125I)EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of (125I)EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound (125I)EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, (125I)EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of (125I)EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of (125I)EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of (125I)EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in (125I)EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.

  6. Cesium cation affinities and basicities

    NASA Astrophysics Data System (ADS)

    Gal, Jean-François; Maria, Pierre-Charles; Massi, Lionel; Mayeux, Charly; Burk, Peeter; Tammiku-Taul, Jaana

    2007-11-01

    This review focuses on the quantitative data related to cesium cation interaction with neutral or negatively charged ligands. The techniques used for measuring the cesium cation affinity (enthalpies, CCA), and cesium cation basicities (Gibbs free energies, CCB) are briefly described. The quantum chemical calculations methods that were specifically designed for the determination of cesium cation adduct structures and the energetic aspects of the interaction are discussed. The experimental results, obtained essentially from mass spectrometry techniques, and complemented by thermochemical data, are tabulated and commented. In particular, the correlations between cesium cation affinities and lithium cation affinities for the various kinds of ligands (rare gases, polyatomic neutral molecules, among them aromatic compounds and negative ions) serve as a basis for the interpretation of the diverse electrostatic modes of interaction. A brief account of some recent analytical applications of ion/molecule reactions with Cs+, as well as other cationization approaches by Cs+, is given.

  7. "Clickable" agarose for affinity chromatography.

    PubMed

    Punna, Sreenivas; Kaltgrad, Eiton; Finn, M G

    2005-01-01

    Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

  8. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  9. The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

    PubMed

    Laver, Derek R; Hamada, Tomoyo; Fessenden, James D; Ikemoto, Noriaki

    2007-12-01

    In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

  10. A multiplexed three-dimensional paper-based electrochemical impedance device for simultaneous label-free affinity sensing of total and glycated haemoglobin: The potential of using a specific single-frequency value for analysis.

    PubMed

    Boonyasit, Yuwadee; Chailapakul, Orawon; Laiwattanapaisal, Wanida

    2016-09-14

    A novel three-dimensional paper-based electrochemical impedance device (3D-PEID) is first introduced for measuring multiple diabetes markers. Herein, a simple 3D-PEID composed of a dual screen-printed electrode on wax-patterned paper coupled with a multilayer of magnetic paper was fabricated for label-free electrochemical detection. The results clearly demonstrated in a step-wise manner that the haptoglobin (Hp)-modified and 3-aminophenylboronic acid (APBA)-modified eggshell membranes (ESMs) were highly responsive to a clinically relevant range of total (0.5-20 g dL(-1); r(2) = 0.989) and glycated haemoglobin (HbA1c) (2.3%-14%; r(2) = 0.997) levels with detection limits (S/N = 3) of 0.08 g dL(-1) and 0.21%, respectively. The optimal binding frequencies of total haemoglobin and HbA1c to their specific recognition elements were 5.18 Hz and 9.99 Hz, respectively. The within-run coefficients of variation (CV) were 1.84%, 2.18%, 1.72%, and 2.01%, whereas the run-to-run CVs were 2.11%, 2.41%, 2.08%, and 2.21%, when assaying two levels of haemoglobin and HbA1c, respectively. The CVs for the haemoglobin and HbA1c levels measured on ten independently fabricated paper-based sheets were 1.96% and 2.10%, respectively. These results demonstrated that our proposed system achieved excellent precision for the simultaneous detection of total haemoglobin and HbA1c, with an acceptable reproducibility of fabrication. The long-term stability of the Hp-modified eggshell membrane (ESM) was 98.84% over a shelf-life of 4 weeks, enabling the possibility of storage or long-distance transport to remote regions, particularly in resource-limited settings; however, for the APBA-modified ESM, the stability was 92.35% over a one-week period. Compared with the commercial automated method, the results demonstrated excellent agreement between the techniques (p-value < 0.05), thus permitting the potential application of 3D-PEID for the monitoring of the glycaemic status in diabetic

  11. A multiplexed three-dimensional paper-based electrochemical impedance device for simultaneous label-free affinity sensing of total and glycated haemoglobin: The potential of using a specific single-frequency value for analysis.

    PubMed

    Boonyasit, Yuwadee; Chailapakul, Orawon; Laiwattanapaisal, Wanida

    2016-09-14

    A novel three-dimensional paper-based electrochemical impedance device (3D-PEID) is first introduced for measuring multiple diabetes markers. Herein, a simple 3D-PEID composed of a dual screen-printed electrode on wax-patterned paper coupled with a multilayer of magnetic paper was fabricated for label-free electrochemical detection. The results clearly demonstrated in a step-wise manner that the haptoglobin (Hp)-modified and 3-aminophenylboronic acid (APBA)-modified eggshell membranes (ESMs) were highly responsive to a clinically relevant range of total (0.5-20 g dL(-1); r(2) = 0.989) and glycated haemoglobin (HbA1c) (2.3%-14%; r(2) = 0.997) levels with detection limits (S/N = 3) of 0.08 g dL(-1) and 0.21%, respectively. The optimal binding frequencies of total haemoglobin and HbA1c to their specific recognition elements were 5.18 Hz and 9.99 Hz, respectively. The within-run coefficients of variation (CV) were 1.84%, 2.18%, 1.72%, and 2.01%, whereas the run-to-run CVs were 2.11%, 2.41%, 2.08%, and 2.21%, when assaying two levels of haemoglobin and HbA1c, respectively. The CVs for the haemoglobin and HbA1c levels measured on ten independently fabricated paper-based sheets were 1.96% and 2.10%, respectively. These results demonstrated that our proposed system achieved excellent precision for the simultaneous detection of total haemoglobin and HbA1c, with an acceptable reproducibility of fabrication. The long-term stability of the Hp-modified eggshell membrane (ESM) was 98.84% over a shelf-life of 4 weeks, enabling the possibility of storage or long-distance transport to remote regions, particularly in resource-limited settings; however, for the APBA-modified ESM, the stability was 92.35% over a one-week period. Compared with the commercial automated method, the results demonstrated excellent agreement between the techniques (p-value < 0.05), thus permitting the potential application of 3D-PEID for the monitoring of the glycaemic status in diabetic

  12. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  13. Effectors of hemoglobin. Separation of allosteric and affinity factors.

    PubMed Central

    Marden, M C; Bohn, B; Kister, J; Poyart, C

    1990-01-01

    The relative contributions of the allosteric and affinity factors toward the change in p50 have been calculated for a series of effectors of hemoglobin (Hb). Shifts in the ligand affinity of deoxy Hb and the values for 50% ligand saturation (p50) were obtained from oxygen equilibrium data. Because the high-affinity parameters (liganded conformation) are poorly determined from the equilibrium curves, they were determined from kinetic measurements of the association and dissociation rates with CO as ligand. The CO on-rates were obtained by flash photolysis measurements. The off-rates were determined from the rate of oxidation of HbCO by ferricyanide, or by replacement of CO with NO. The partition function of fully liganded hemoglobin for oxygen and CO is only slightly changed by the effectors. Measurements were made in the presence of the effectors 2,3-diphosphoglycerate (DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), and two recently synthesized derivatives of Bzf (LR16 and L35). Values of p50 change by over a factor of 60; the on-rates decrease by nearly a factor of 8, with little change in the off-rates for the liganded conformation. The data indicate that both allosteric and affinity parameters are changed by the effectors; the changes in ligand affinity represent the larger contribution toward shifts in p50. PMID:2306490

  14. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  15. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  16. Two-parameter twisted quantum affine algebras

    NASA Astrophysics Data System (ADS)

    Jing, Naihuan; Zhang, Honglian

    2016-09-01

    We establish Drinfeld realization for the two-parameter twisted quantum affine algebras using a new method. The Hopf algebra structure for Drinfeld generators is given for both untwisted and twisted two-parameter quantum affine algebras, which include the quantum affine algebras as special cases.

  17. Binding of dopamine and 3-methoxytyramine as l-DOPA metabolites to human alpha(2)-adrenergic and dopaminergic receptors.

    PubMed

    Alachkar, Amal; Brotchie, Jonathan M; Jones, Owen T

    2010-07-01

    The ability of l-3,4-dihydroxyphenylalanine (l-DOPA), l-DOPA-methyl ester and their major metabolites, dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic (HVA), 3-O-methyldopa and 3-methoxytyramine (3-MT) to bind to alpha(2) adrenergic and D1 and D2 dopamine receptors was assessed by radioligand binding to cloned human receptors expressed in cell lines. As anticipated, dopamine bound with high affinity to D1 (IC(50) 1.1 + or - 0.16 microM) and D2 (IC(50) 0.7 + or - 0.3 microM) dopamine receptors. However, dopamine also bound with high affinity to alpha(2A) (IC(50) was 2.6 + or - 0.5 microM), alpha(2C) (IC(50) 3.2 + or - 0.7 microM). 3-MT bound to alpha(2A) with high affinity (IC(50), 3.6 + or - 0.2 microM) though moderate affinity to alpha(2)c, D1 and D2 receptors (values of IC(50) were 55 + or - 14, 121 + or - 43, 36 + or - 14 microM, respectively). l-DOPA-methyl ester bound with high affinity to alpha(2) (IC(50) 17-36 microM) but not dopamine receptors (IC(50) 0.9-2.5 mM). l-DOPA, 3-O-methyldopa and DOPAC had no observable effect on binding to any of the receptors tested. These data suggest that the effects of l-DOPA in Parkinson's disease may result from actions of its metabolites dopamine and 3-MT on both dopaminergic and non-dopaminergic receptors. These findings may provide explanations for the differences between l-DOPA and dopamine receptor agonists in mediating anti-parkinsonian effects and propensity to be associated with dyskinesia and motor complications such as wearing-off and on-off.

  18. [The effect of blood serum polyreactive immunoglobulins on serum antibody affinity determination].

    PubMed

    Bobrovnik, S A; Demchenko, M A; Komisarenko, S V

    2010-01-01

    The presence of polyreactive immunoglobulins in sera may substantially influence on the accuracy of antibody affinity determination. In order to obtain precise values of antibody affinity one should apply one of two following ways. First, one should block polyreactive immunoglobulins with high concentration of Twin 20 and high concentration of any antigen which does not interact with studying antibody. After this antibody affinity may be determined by traditional methods. Another way is the application of the method suggested by us earlier, which allow determining affinity of two antibodies in a mixture and the relation of their concentrations.

  19. Pharmacological characterization of α-elapitoxin-Al2a from the venom of the Australian pygmy copperhead (Austrelaps labialis): an atypical long-chain α-neurotoxin with only weak affinity for α7 nicotinic receptors.

    PubMed

    Marcon, Francesca; Leblanc, Mathieu; Vetter, Irina; Lewis, Richard J; Escoubas, Pierre; Nicholson, Graham M

    2012-09-15

    Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead (Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77 Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve-muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA(2) value of 7.902 (K(B) = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC(50) of 1.2 μM, and failed to inhibit ganglionic α3β2/α3β4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)(2)βγδ nAChRs it lacks the key Ala(28) and Arg(36) residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation. PMID:22771828

  20. Affinity selection-based two-dimensional chromatography coupled with high-performance liquid chromatography-mass spectrometry for discovering xanthine oxidase inhibitors from Radix Salviae Miltiorrhizae.

    PubMed

    Fu, Yu; Mo, Hua-Yan; Gao, Wen; Hong, Jia-Ying; Lu, Jun; Li, Ping; Chen, Jun

    2014-08-01

    Xanthine oxidase (XOD) is a key oxidative enzyme to the pathogenesis of hyperuricemia and certain diseases induced by excessive reactive oxygen species. XOD inhibitors could provide an important therapeutic approach to treat such diseases. A new method using affinity selection-based two-dimensional chromatography coupled with liquid chromatography-mass spectrometry was developed for the online screening of potential XOD inhibitors from Radix Salviae Miltiorrhizae. Based on our previous study, the two-dimensional, turbulent-flow chromatography (TFC) was changed to a mixed-mode anion-exchange/reversed-phase column and one reversed-phase column. The developed method was validated to be selective and sensitive for screening XOD-binding compounds, especially weak acidic ones, in the extracts. Three salvianolic acids were screened from the Radix Salviae Miltiorrhizae extract via the developed method. The XOD inhibitory activities of salvianolic acid C and salvianolic acid A were confirmed, and their inhibitory modes were measured. Salvianolic acid C exhibited potent XOD inhibitory activity with an IC(50) of 9.07 μM. This work demonstrated that the developed online, two-dimensional TFC/LC-MS method was effective in discovering the binding affinity of new compounds from natural extracts for target proteins, even at low concentrations.

  1. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain.

    PubMed

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10(-10) M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  2. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    PubMed Central

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  3. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  4. Engineering antibody affinity and specificity.

    PubMed

    Webster, D M; Roberts, S; Cheetham, J C; Griest, R; Rees, A R

    1988-01-01

    A combination of ab initio calculations, "knowledge-based prediction", molecular graphics and site-directed mutagenesis has enabled us to probe the molecular details of antibody:antigen recognition and binding and to alter the affinity and specificity of an antibody for its antigen. The significance of electrostatic hydrogen bonding, hydrophilic/hydrophobic patch matching and van der Waals interactions as well as CDR:CDR interactions are discussed in relation to the results of site-directed mutagenesis experiments on the anti-lysozyme antibody Gloop2. The ability to generate reconstructed antibodies, chimeric antibodies, catalytic antibodies and the use of modelled antibodies for the design of drugs is discussed. PMID:3209295

  5. Proton affinity of methyl nitrate - Less than proton affinity of nitric acid

    NASA Technical Reports Server (NTRS)

    Lee, Timothy J.; Rice, Julia E.

    1992-01-01

    Several state-of-the-art ab initio quantum mechanical methods were used to investigate the equilibrium structure, dipole moments, harmonic vibrational frequencies, and IR intensities of methyl nitrate, methanol, and several structures of protonated methyl nitrate, using the same theoretical methods as in an earlier study (Lee and Rice, 1992) of nitric acid. The ab initio results for methyl nitrate and methanol were found to be in good agreement with available experimental data. The proton affinity (PA) of methyl nitrate was calculated to be 176.9 +/-5 kcal/mol, in excellent agreement with the experimental value 176 kcal/mol obtained by Attina et al. (1987) and less than the PA value of nitric acid. An explanation of the discrepancy of the present results with those of an earlier study on protonated nitric acid is proposed.

  6. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  7. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, K.S.

    1996-04-01

    Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

  8. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  9. Non-affine deformations in polymer hydrogels

    PubMed Central

    Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

    2012-01-01

    Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation. PMID:23002395

  10. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  11. Affinity functions: recognizing essential parameters in fuzzy connectedness based image segmentation

    NASA Astrophysics Data System (ADS)

    Ciesielski, Krzysztof C.; Udupa, Jayaram K.

    2009-02-01

    Fuzzy connectedness (FC) constitutes an important class of image segmentation schemas. Although affinity functions represent the core aspect (main variability parameter) of FC algorithms, they have not been studied systematically in the literature. In this paper, we present a thorough study to fill this gap. Our analysis is based on the notion of equivalent affinities: if any two equivalent affinities are used in the same FC schema to produce two versions of the algorithm, then these algorithms are equivalent in the sense that they lead to identical segmentations. We give a complete characterization of the affinity equivalence and show that many natural definitions of affinity functions and their parameters used in the literature are redundant in the sense that different definitions and values of such parameters lead to equivalent affinities. We also show that two main affinity types - homogeneity based and object feature based - are equivalent, respectively, to the difference quotient of the intensity function and Rosenfeld's degree of connectivity. In addition, we demonstrate that any segmentation obtained via relative fuzzy connectedness (RFC) algorithm can be viewed as segmentation obtained via absolute fuzzy connectedness (AFC) algorithm with an automatic and adaptive threshold detection. We finish with an analysis of possible ways of combining different component affinities that result in non equivalent affinities.

  12. Secondary amine analogues of 3 beta-(4'-substituted phenyl)tropane-2 beta-carboxylic acid esters and N-norcocaine exhibit enhanced affinity for serotonin and norepinephrine transporters.

    PubMed

    Boja, J W; Kuhar, M J; Kopajtic, T; Yang, E; Abraham, P; Lewin, A H; Carroll, F I

    1994-04-15

    N-Norcocaine (2) and six N-nor-3 beta-(4'-substituted phenyl)tropane-2 beta-carboxylic acid esters (4a-f) were synthesized by N-demethylation of cocaine (1) and the appropriate 3 beta-(substituted phenyl)-tropane analogues (3a-f) with alpha-chloroethyl chloroformate. Radioligand binding affinities of 2 and 4a-f at the DA, 5-HT, and NE transporter were measured and compared to those of 1 and 3a-f. N-Demethylation produced relatively small effects at the DA transporter. In contrast, 4-19-fold and 2-44-fold enhanced affinity at the serotonin and norepinephrine transporter resulted from demethylation. N-Nor-3 beta-(4'-iodophenyl)tropane-2 beta-carboxylic acid methyl ester (4d) with an IC50 = 0.36 nM showed the greatest affinity for the serotonin transporter. However, N-nor-3 beta-(4'-ethylphenyl)tropane-2 beta-carboxylic acid methyl ester (4e) showed the greatest selectivity for the serotonin transporter.

  13. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  14. Feature Matching with Affine-Function Transformation Models.

    PubMed

    Li, Hongsheng; Huang, Xiaolei; Huang, Junzhou; Zhang, Shaoting

    2014-12-01

    Feature matching is an important problem and has extensive uses in computer vision. However, existing feature matching methods support either a specific or a small set of transformation models. In this paper, we propose a unified feature matching framework which supports a large family of transformation models. We call the family of transformation models the affine-function family, in which all transformations can be expressed by affine functions with convex constraints. In this framework, the goal is to recover transformation parameters for every feature point in a template point set to calculate their optimal matching positions in an input image. Given pairwise feature dissimilarity values between all points in the template set and the input image, we create a convex dissimilarity function for each template point. Composition of such convex functions with any transformation model in the affine-function family is shown to have an equivalent convex optimization form that can be optimized efficiently. Four example transformation models in the affine-function family are introduced to show the flexibility of our proposed framework. Our framework achieves 0.0 percent matching errors for both CMU House and Hotel sequences following the experimental setup in [6]. PMID:26353148

  15. Methods for Improving Aptamer Binding Affinity.

    PubMed

    Hasegawa, Hijiri; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. PMID:27043498

  16. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  17. Multiple GPCR conformations and signalling pathways: implications for antagonist affinity estimates

    PubMed Central

    Baker, Jillian G.; Hill, Stephen J.

    2007-01-01

    Antagonist affinity measurements have traditionally been considered important in characterizing the cell-surface receptors present in a particular cell or tissue. A central assumption has been that antagonist affinity is constant for a given receptor–antagonist interaction, regardless of the agonist used to stimulate that receptor or the downstream response that is measured. As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes. Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions. Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured). PMID:17629959

  18. Phytochemical constituents, nutritional values, phenolics, flavonols, flavonoids, antioxidant and cytotoxicity studies on Phaleria macrocarpa (Scheff.) Boerl fruits

    PubMed Central

    2014-01-01

    Background The edible fruits of Phaleria macrocarpa (Scheff.) Boerl are widely used in traditional medicine in Indonesia. It is used to treat a variety of medical conditions such as - cancer, diabetes mellitus, allergies, liver and heart diseases, kidney failure, blood diseases, high blood pressure, stroke, various skin diseases, itching, aches, and flu. Therefore, it is of great interest to determine the biochemical and cytotoxic properties of the fruit extracts. Methods The methanol, hexane, chloroform, ethyl acetate, and water extracts of P. macrocarpa fruits were examined for phytochemicals, physicochemicals, flavonols, flavonoids and phenol content. Its nutritional value (A.O.A.C method), antioxidant properties (DPPH assay) and cytotoxicity (MTT cell proliferation assay) were also determined. Results A preliminary phyotochemical screening of the different crude extracts from the fruits of P. macrocarpa showed the presence secondary metabolites such as of flavonoids, phenols, saponin glycosides and tannins. The ethyl acetate and methanol extracts displayed high antioxidant acitivity (IC50 value of 8.15±0.02 ug/mL) in the DPPH assay comparable to that of the standard gallic acid (IC50 value of 10.8±0.02 ug/mL). Evaluation of cytotoxic activity showed that the crude methanol extract possessed excellent anti-proliferative activity against SKOV-3 (IC50 7.75±2.56 μg/mL) after 72 hours of treatment whilst the hexane and ethyl acetate extracts displayed good cytotoxic effect against both SKOV-3 and MDA-MB231 cell lines. The chloroform extract however, showed selective inhibitory activity in the breast cancer cell line MDA-MB231 (IC50 7.80±1.57 μg/mL) after 48 hours of treatment. There was no cytotoxic effect observed in the Ca Ski cell line and the two normal cell lines (MRC-5 and WRL-68). Conclusion The methanol extract and the ethyl acetate fraction of P. macrocarpa fruits exhibited good nutritional values, good antioxidant and cytotoxic activities, and merits

  19. Multiplexed protein profiling by sequential affinity capture

    PubMed Central

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter

    2016-01-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off‐target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi‐automated sequential capture assay. This novel bead‐based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read‐out by a secondary capture bead array. We demonstrate in a proof‐of‐concept setting that target detection via two sequential affinity interactions reduced off‐target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA‐based signal amplification, and demonstrate the applicability of the dual capture bead‐based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  20. Semiempirical Theories of the Affinities of Negative Atomic Ions

    NASA Technical Reports Server (NTRS)

    Edie, John W.

    1961-01-01

    The determination of the electron affinities of negative atomic ions by means of direct experimental investigation is limited. To supplement the meager experimental results, several semiempirical theories have been advanced. One commonly used technique involves extrapolating the electron affinities along the isoelectronic sequences, The most recent of these extrapolations Is studied by extending the method to Include one more member of the isoelectronic sequence, When the results show that this extension does not increase the accuracy of the calculations, several possible explanations for this situation are explored. A different approach to the problem is suggested by the regularities appearing in the electron affinities. Noting that the regular linear pattern that exists for the ionization potentials of the p electrons as a function of Z, repeats itself for different degrees of ionization q, the slopes and intercepts of these curves are extrapolated to the case of the negative Ion. The method is placed on a theoretical basis by calculating the Slater parameters as functions of q and n, the number of equivalent p-electrons. These functions are no more than quadratic in q and n. The electron affinities are calculated by extending the linear relations that exist for the neutral atoms and positive ions to the negative ions. The extrapolated. slopes are apparently correct, but the intercepts must be slightly altered to agree with experiment. For this purpose one or two experimental affinities (depending on the extrapolation method) are used in each of the two short periods. The two extrapolation methods used are: (A) an isoelectronic sequence extrapolation of the linear pattern as such; (B) the same extrapolation of a linearization of this pattern (configuration centers) combined with an extrapolation of the other terms of the ground configurations. The latter method Is preferable, since it requires only experimental point for each period. The results agree within

  1. Estimation of human blood LC50 values for use in modeling of in vitro-in vivo data of the ACuteTox project.

    PubMed

    Sjöström, Michael; Kolman, Ada; Clemedson, Cecilia; Clothier, Richard

    2008-08-01

    The main aim of the ACuteTox project, under EU 6th Framework programme, is to investigate whether animal toxicity tests for acute systemic toxicity could be replaced by a combination of alternative assays. Data for 97 reference chemicals was collected in the ACuteTox database (Acutoxbase), designed to handle invitro and invivo (human and animal) lodged data. The principal basis for demonstration of the applicability of invitro tests is the invitro-invivo modeling, by using statistical correlation between invitro IC50 molar values (the 50% inhibitory concentration for the endpoints measured) and human blood molar concentrations LC50 (50% lethal concentrations). The LC50 values were calculated from time-related sub-lethal and lethal blood concentrations determined from human acute poisoning cases. The 3T3 standard NRU assay (3T3 NRU) was chosen, among the various basal cytotoxicity assays, applied in the ACuteTox project, to demonstrate the applicability of the IC50/LC50 values for invitro-invivo modeling. Linear regression analysis between IC50 (x) and LC50 (y) gave an explained variance R2=0.56 for the 67 reference chemicals, for which both sets of data were available. The results demonstrated usefulness of human LC50 values for invitro-invivo evaluation of the predictability of basal cytotoxicity assays for human acute systemic toxicity. The R2 value of 0.56 shows, as in the MEIC study, that additional organ-specific and biokinetic tests are needed in order to improve the predictability.

  2. Affinity Proteomics in the mountains: Alpbach 2015.

    PubMed

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167

  3. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  4. Isotope shift in the electron affinity of lithium

    SciTech Connect

    Bubin, Sergiy; Komasa, Jacek; Stanke, Monika; Adamowicz, Ludwik

    2009-12-21

    Very accurate electron affinity (EA) calculations of {sup 6}Li and {sup 7}Li (and {sup {infinity}L}i) have been performed using explicitly correlated Gaussian functions and a variational approach that explicitly includes the nuclear motion in the calculations (i.e., the approach that does not assume the Born-Oppenheimer approximation). The leading relativistic and quantum electrodynamics corrections to the electron affinities were also calculated. The results are the most accurate theoretical values obtained for the studied systems to date. Our best estimates of the {sup 7}Li and {sup 6}Li EAs are 4984.9842(30) and 4984.9015(30) cm{sup -1}, respectively, and of the {sup 7}Li/{sup 6}Li EA isotope shift is 0.0827 cm{sup -1}.

  5. Isotope shift in the electron affinity of lithium.

    PubMed

    Bubin, Sergiy; Komasa, Jacek; Stanke, Monika; Adamowicz, Ludwik

    2009-12-21

    Very accurate electron affinity (EA) calculations of (6)Li and (7)Li (and (infinity)Li) have been performed using explicitly correlated Gaussian functions and a variational approach that explicitly includes the nuclear motion in the calculations (i.e., the approach that does not assume the Born-Oppenheimer approximation). The leading relativistic and quantum electrodynamics corrections to the electron affinities were also calculated. The results are the most accurate theoretical values obtained for the studied systems to date. Our best estimates of the (7)Li and (6)Li EAs are 4984.9842(30) and 4984.9015(30) cm(-1), respectively, and of the (7)Li/(6)Li EA isotope shift is 0.0827 cm(-1).

  6. Molecular modeling of oscillating GHz electric field influence on the kinesin affinity to microtubule

    NASA Astrophysics Data System (ADS)

    R. Saeidi, H.; S. Setayandeh, S.; Lohrasebi, A.

    2015-08-01

    Kinesin is a microtubule-associated motor protein which can respond to the external electric field due to its polarity. Using a molecular dynamics simulation method, the effect of such a field on the affinity of kinesin to the αβ-tubulin is investigated in this study. To consider kinesin affinity, the system is exposed to an electric field of 0.03 V/nm with frequency values of 1, 2, …, 9, and 10 GHz. It is found that the applied electric field can change kinesin affinity to the microtubule. These changes could perturb the normal operation of kinesin, such as the processive motility of kinesin on the microtubule.

  7. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  8. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  9. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  10. Designing Chaotic Systems by Piecewise Affine Systems

    NASA Astrophysics Data System (ADS)

    Wu, Tiantian; Li, Qingdu; Yang, Xiao-Song

    Based on mathematical analysis, this paper provides a methodology to ensure the existence of homoclinic orbits in a class of three-dimensional piecewise affine systems. In addition, two chaotic generators are provided to illustrate the effectiveness of the method.

  11. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  12. Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa.

    PubMed Central

    Yerima, A; Safi, A; Gastin, I; Michalski, J C; Saunier, M; Gueant, J L

    1996-01-01

    We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor. PMID:8573109

  13. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  14. MCDHF calculation of electron affinities of Group I and Group IB atomic anions

    NASA Astrophysics Data System (ADS)

    Li, Junqin; Zhao, Zilong; Zhang, Xuemei

    2014-08-01

    The affinities of negative ions for elements of Group I and Group IB have been calculated using the multi-configuration Dirac-Hartree-Fock (MCDHF) method. The difference between the total energy of the ground state of the atom and that of its anion is used to obtain the electron affinity. The theoretical results for these elements agree well with measured values, and have a deviation less than 0.5% with respect to measured values for most of the elements. With a systematic calculation method, this work gives a high-accuracy theoretical value for the electron affinities of the elements of Group I and Group IB. For element Fr, there is no experimental value.

  15. Heteroaromatic analogs of 1-[2-(diphenylmethoxy)ethyl]- and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909) as high-affinity dopamine reuptake inhibitors.

    PubMed

    Matecka, D; Lewis, D; Rothman, R B; Dersch, C M; Wojnicki, F H; Glowa, J R; DeVries, A C; Pert, A; Rice, K C

    1997-02-28

    A new series of heteroaromatic GBR 12935 [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine] (I) and GBR 12909 [1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine] (2) analogs was synthesized and evaluated as dopamine transporter (DAT) ligands. Analogs 5-16, in which the benzene ring in the phenylpropyl side chain of the GBR molecule had been replaced with a thiophene, furan, or pyridine ring, exhibited high affinity and selectivity for the DAT vs serotonin transporter (SERT) and stimulated locomotor activity in rats in a manner similar to the parent compound 2. In cocaine and food self-administration studies in rhesus monkeys, both thiophene-containing (6 and 8) and pyridine-containing (14 and 16) derivatives displayed potency comparable to 2 in decreasing the cocaine-maintained responding at the doses tested (0.8, 1.7, and 3 mg/kg). However, these compounds did not produce the degree of separation between food- and cocaine-maintained responding that was seen with 2. Among the bicyclic fused-ring congeners 17-38, the indole-containing analog of 2, 22, showed the greatest affinity for binding to the DAT, with IC50 = 0.7 nM, whereas the corresponding indole-containing derivative of 1, 21, displayed the highest selectivity (over 600-fold) at this site vs the SERT site.

  16. Affinity learning with diffusion on tensor product graph.

    PubMed

    Yang, Xingwei; Prasad, Lakshman; Latecki, Longin Jan

    2013-01-01

    In many applications, we are given a finite set of data points sampled from a data manifold and represented as a graph with edge weights determined by pairwise similarities of the samples. Often the pairwise similarities (which are also called affinities) are unreliable due to noise or due to intrinsic difficulties in estimating similarity values of the samples. As observed in several recent approaches, more reliable similarities can be obtained if the original similarities are diffused in the context of other data points, where the context of each point is a set of points most similar to it. Compared to the existing methods, our approach differs in two main aspects. First, instead of diffusing the similarity information on the original graph, we propose to utilize the tensor product graph (TPG) obtained by the tensor product of the original graph with itself. Since TPG takes into account higher order information, it is not a surprise that we obtain more reliable similarities. However, it comes at the price of higher order computational complexity and storage requirement. The key contribution of the proposed approach is that the information propagation on TPG can be computed with the same computational complexity and the same amount of storage as the propagation on the original graph. We prove that a graph diffusion process on TPG is equivalent to a novel iterative algorithm on the original graph, which is guaranteed to converge. After its convergence we obtain new edge weights that can be interpreted as new, learned affinities. We stress that the affinities are learned in an unsupervised setting. We illustrate the benefits of the proposed approach for data manifolds composed of shapes, images, and image patches on two very different tasks of image retrieval and image segmentation. With learned affinities, we achieve the bull's eye retrieval score of 99.99 percent on the MPEG-7 shape dataset, which is much higher than the state-of-the-art algorithms. When the data

  17. Synthesis and NMDA receptor affinity of fluorinated dioxadrol analogues.

    PubMed

    Banerjee, Ashutosh; Schepmann, Dirk; Wünsch, Bernhard

    2010-06-01

    A series of dioxadrol analogues with fluorine substituents in position 4 of the piperidine ring has been synthesized and pharmacologically evaluated. The key step in the synthesis was the fluorination of diastereomeric piperidones 6a and 6c as well as diastereomeric alcohols 9a and 9c with DAST. The reaction of the alcohols 9a and 9c took place with inversion of configuration. After removal of the Cbz-protective group, the NMDA receptor affinities of the resulting secondary amines 8a, 8c, 12b, and 12d were investigated in receptor binding studies. It was shown that the like-configuration of the ring junction was crucial for high NMDA receptor affinity. An axially oriented fluorine atom in position 4 led to 2-(2,2-diphenyl-1,3-dioxolan-4-yl)-4-fluoropiperidine (12d, WMS-2517) with a K(i)-value of 27nM. The NMDA receptor affinity of 8c (WMS-2513) with an additional fluorine atom in equatorial 4-position was slightly reduced (K(i)=81 nM). Both fluorinated dioxadrol derivatives 8c and 12d showed high selectivity against sigma(1) and sigma(2) receptors as well as the polyamine binding site of NR2B receptors.

  18. Proton Affinity of Isomeric Dipeptides Containing Lysine and Non-Proteinogenic Lysine Homologues.

    PubMed

    Batoon, Patrick; Ren, Jianhua

    2016-08-18

    Conformational effects on the proton affinity of oligopeptides have been studied using six alanine (A)-based acetylated dipeptides containing a basic probe that is placed closest to either the C- or the N-terminus. The basic probe includes Lysine (Lys) and two nonproteinogenic Lys-homologues, ornithine (Orn) and 2,3-diaminopropionic acid (Dap). The proton affinities of the peptides have been determined using the extended Cooks kinetic method in a triple quadrupole mass spectrometer. Computational studies have been carried out to search for the lowest energy conformers and to calculate theoretical proton affinities as well as various molecular properties using the density functional theory. The dipeptides containing a C-terminal probe, ALys, AOrn, and ADap, were determined to have a higher proton affinity by 1-4 kcal/mol than the corresponding dipeptides containing an N-terminal probe, LysA, OrnA, and DapA. For either the C-probe peptides or the N-probe peptides, the proton affinity reduces systematically as the side-chain of the probe residue is shortened. The difference in the proton affinities between isomeric peptides is largely associated with the variation of the conformations. The peptides with higher values of the proton affinity adopt a relatively compact conformation such that the protonated peptides can be stabilized through more efficient internal solvation. PMID:27459294

  19. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  20. BC(50): a generalized, unifying affinity descriptor.

    PubMed

    Vacca, Alberto; Francesconi, Oscar; Roelens, Stefano

    2012-12-01

    Assessing binding affinities is an unavoidable step that we come across any time interactions between binding species are investigated. A quantitative evaluation of binding affinities relies on the determination of binding constants but, whilst the binding constant fully defines the affinity of a reagent for a ligand when only one complex species is formed, the same is not true when the interacting partners form more than one complex of different stoichiometry, because all complexes contribute to the overall binding affinity. Unfortunately, this situation is the rule rather than the exception in chemical systems, but a generally accepted solution for this issue has not yet been settled. In this Personal Account, we describe the evolution, from the initial idea to a fully developed stage, of a binding descriptor that has been developed with the aim of filling this gap, thereby providing scientists in all fields of chemistry with a unifying tool for the assessment of binding affinities based on the knowledge of the binding constants in systems that involve any number of complex species.

  1. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  2. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  3. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  4. Valuing Essays: Essaying Values

    ERIC Educational Resources Information Center

    Badley, Graham

    2010-01-01

    The essay regularly comes under attack. It is criticised for being rigidly linear rather than flexible and reflective. I first challenge this view by examining reasons why the essay should be valued as an important genre. Secondly, I propose that in using the essay form students and academics necessarily exemplify their own critical values. Essays…

  5. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same time,…

  6. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    ERIC Educational Resources Information Center

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  7. Affinity enhancement by dendritic side chains in synthetic carbohydrate receptors.

    PubMed

    Destecroix, Harry; Renney, Charles M; Mooibroek, Tiddo J; Carter, Tom S; Stewart, Patrick F N; Crump, Matthew P; Davis, Anthony P

    2015-02-01

    Dendritic side chains have been used to modify the binding environment in anthracene-based synthetic carbohydrate receptors. Control of length, charge, and branching enabled the positioning of side-chain carboxylate groups in such a way that they assisted in binding substrates rather than blocking the cavity. Conformational degeneracy in the dendrimers resulted in effective preorganization despite the flexibility of the system. Strong binding was observed to glucosammonium ions in water, with Ka values up to 7000 M(-1) . Affinities for uncharged substrates (glucose and N-acetylglucosamine) were also enhanced, despite competition from solvent and the absence of electrostatic interactions. PMID:25645064

  8. Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

    PubMed Central

    Moon, Seung Kee; Park, So Ra; Park, Ami; Oh, Hyun Mi; Shin, Hyun Jung; Jeon, Eun Ju; Kim, Seiwhan; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je

    2016-01-01

    To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5. PMID:26743905

  9. Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen.

    PubMed

    Moon, Seung Kee; Park, So Ra; Park, Ami; Oh, Hyun Mi; Shin, Hyun Jung; Jeon, Eun Ju; Kim, Seiwhan; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je

    2016-03-01

    To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.

  10. Chemically Modified DNA Aptamers Bind Interleukin-6 with High Affinity and Inhibit Signaling by Blocking Its Interaction with Interleukin-6 Receptor

    PubMed Central

    Gupta, Shashi; Hirota, Masao; Waugh, Sheela M.; Murakami, Ikuo; Suzuki, Tomoki; Muraguchi, Masahiro; Shibamori, Masafumi; Ishikawa, Yuichi; Jarvis, Thale C.; Carter, Jeffrey D.; Zhang, Chi; Gawande, Bharat; Vrkljan, Michael; Janjic, Nebojsa; Schneider, Daniel J.

    2014-01-01

    Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2′-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases. PMID:24415766

  11. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  12. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  13. European and international collaboration in affinity proteomics.

    PubMed

    Stoevesandt, Oda; Taussig, Michael J

    2012-06-15

    In affinity proteomics, specific protein-binding molecules (a.k.a. binders), principally antibodies, are applied as reagents in proteome analysis. In recent years, advances in binder technologies have created the potential for an unprecedented view on protein expression and distribution patterns in plasma, cells and tissues and increasingly on protein function. Particular strengths of affinity proteomics methods include detecting proteins in their natural environments of cell or tissue, high sensitivity and selectivity for detection of low abundance proteins and exploiting binding actions such as functional interference in living cells. To maximise the use and impact of affinity reagents, it will be essential to create comprehensive, standardised binder collections. With this in mind, the EU FP7 programme AFFINOMICS (http://www.affinomics.org), together with the preceding EU programmes ProteomeBinders and AffinityProteome, aims to extend affinity proteomics research by generating a large-scale resource of validated protein-binding molecules for characterisation of the human proteome. Activity is directed at producing binders to about 1000 protein targets, primarily in signal transduction and cancer, by establishing a high throughput, coordinated production pipeline. An important aspect of AFFINOMICS is the development of highly efficient recombinant selection methods, based on phage, cell and ribosome display, capable of producing high quality binders at greater throughput and lower cost than hitherto. The programme also involves development of innovative and sensitive technologies for specific detection of target proteins and their interactions, and deployment of binders in proteomics studies of clinical relevance. The need for such binder generation programmes is now recognised internationally, with parallel initiatives in the USA for cancer (NCI) and transcription factors (NIH) and within the Human Proteome Organisation (HUPO). The papers in this volume of New

  14. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  15. The dynamics of metric-affine gravity

    SciTech Connect

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-05-15

    Highlights: > The role and the dynamics of the connection in metric-affine theories is explored. > The most general second order action does not lead to a dynamical connection. > Including higher order invariants excites new degrees of freedom in the connection. > f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy theories to

  16. Affine Invariant Character Recognition by Progressive Removing

    NASA Astrophysics Data System (ADS)

    Iwamura, Masakazu; Horimatsu, Akira; Niwa, Ryo; Kise, Koichi; Uchida, Seiichi; Omachi, Shinichiro

    Recognizing characters in scene images suffering from perspective distortion is a challenge. Although there are some methods to overcome this difficulty, they are time-consuming. In this paper, we propose a set of affine invariant features and a new recognition scheme called “progressive removing” that can help reduce the processing time. Progressive removing gradually removes less feasible categories and skew angles by using multiple classifiers. We observed that progressive removing and the use of the affine invariant features reduced the processing time by about 60% in comparison to a trivial one without decreasing the recognition rate.

  17. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  18. Adsorption affinity of anions on metal oxyhydroxides

    NASA Astrophysics Data System (ADS)

    Pechenyuk, S. I.; Semushina, Yu. P.; Kuz'mich, L. F.

    2013-03-01

    The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO{4/3-}, CO{3/2-} > C2O{4/2-}, C(OH)(CH2)2(COO){3/3-}, (CHOH)2(COO){2/2-} > CrO{4/2-} ≫ SO{4/2-}.

  19. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  20. Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase alpha and a new carrier for inhibitor-affinity chromatography.

    PubMed

    Mizushina, Yoshiyuki; Ishidoh, Tomomi; Kamisuki, Shinji; Nakazawa, Satoshi; Takemura, Masaharu; Sugawara, Fumio; Yoshida, Hiromi; Sakaguchi, Kengo

    2003-02-01

    Two flavonoid glycosides, kaempferol 3-O-(6"-acetyl)-beta-glucopyranoside (KAG) and quercetin 3-O-(6"-acetyl)-beta-glucopyranoside (QAG), were found to be inhibitors of eukaryotic DNA polymerases from a Japanese vegetable, Petasites japonicus. These compounds inhibited the activities of mammalian replicative DNA polymerases (i.e., pol alpha, delta, and epsilon), but not other pol beta, eta, kappa, and lambda activities. KAG was a stronger inhibitor and more selective to pol alpha than QAG. The IC(50) values of KAG for pol alpha, delta, and epsilon were 41, 164, and 127 microM, respectively. The pol alpha inhibition by KAG was non-competitive with respect to both the DNA template-primer and the dNTP substrate. KAG and QAG did not influence the activities of prokaryotic DNA polymerases or other mammalian DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, human telomerase, human DNA topoisomerase I and II, T7 RNA polymerase, and bovine deoxyribonuclease I. Therefore, we concluded that these flavonoid glycosides are moderate replicative DNA polymerase inhibitors leaning more relatively to pol alpha, and could be used as chromatographic carriers to purify the DNA polymerases rather than cytotoxic agents. We then made a KAG-conjugated column such as the epoxy-activated Sepharose 6B. In the column, pol alpha was selectively adsorbed and eluted. PMID:12565887

  1. Interpolation method for accurate affinity ranking of arrayed ligand-analyte interactions.

    PubMed

    Schasfoort, Richard B M; Andree, Kiki C; van der Velde, Niels; van der Kooi, Alex; Stojanović, Ivan; Terstappen, Leon W M M

    2016-05-01

    The values of the affinity constants (kd, ka, and KD) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (kd and ka) and dissociation equilibrium (KD) constants for various ligand densities and analyte concentrations are exponentially interpolated to the KD at Rmax = 100 RU response level (KD(R100)).

  2. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  3. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  4. Two bradykinin binding sites with picomolar affinities

    SciTech Connect

    Manning, D.C.; Vavrek, R.; Stewart, J.M.; Snyder, S.H.

    1986-05-01

    Bradykinin (BK) and related peptides exert a wide range of effects on several organ systems. We have attempted to sort out these effects by studying the binding interaction of (/sup 3/H)BK at the membrane level with in vitro receptor binding techniques. High specific activity (/sup 3/H)BK and an enzyme inhibitor cocktail has enabled us to label two BK binding sites with different affinity and peptide specificity in several guinea-pig tissues. In the guinea-pig ileum the high-affinity site has an equilibrium dissociation constant (Kd) for (/sup 3/H)BK of 13 pM and a maximal number of binding sites of 8.3 pmol/g of tissue wet weight. The low-affinity guinea-pig ileum site displays a Kd of 910 pM, a maximum number of binding sites of 14 pmol/g of tissue wet weight and shows a greater selectivity for BK analogs over Lysyl-BK analogs. Two similar sites can also be discriminated in kidney and heart. The potencies of a series of BK analogs at the high-affinity guinea-pig ileum site correlate well with their potencies in contracting ileal smooth muscle. The binding of (/sup 3/H)BK in the guinea-pig ileum is inhibited by physiological concentrations of monovalent and divalent cations.

  5. Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

    PubMed

    Bishop, S A; Bailey, M; Lucke, V M; Stokes, C R

    1992-07-01

    An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an

  6. Methyl cation affinities of neutral and anionic maingroup-element hydrides: trends across the periodic table and correlation with proton affinities.

    PubMed

    Mulder, R Joshua; Guerra, Célia Fonseca; Bickelhaupt, F Matthias

    2010-07-22

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and corresponding entropies) at 298 K of all anionic (XH(n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (i.e., group 18) along the periods 2-6. The cation affinity of the bases decreases from H(+) to CH(3)(+). To understand this trend, we have carried out quantitative bond energy decomposition analyses (EDA). Quantitative correlations are established between the MCA and PA values.

  7. Methyl cation affinities of neutral and anionic maingroup-element hydrides: trends across the periodic table and correlation with proton affinities.

    PubMed

    Mulder, R Joshua; Guerra, Célia Fonseca; Bickelhaupt, F Matthias

    2010-07-22

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and corresponding entropies) at 298 K of all anionic (XH(n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (i.e., group 18) along the periods 2-6. The cation affinity of the bases decreases from H(+) to CH(3)(+). To understand this trend, we have carried out quantitative bond energy decomposition analyses (EDA). Quantitative correlations are established between the MCA and PA values. PMID:20575582

  8. What Value "Value Added"?

    ERIC Educational Resources Information Center

    Richards, Andrew

    2015-01-01

    Two quantitative measures of school performance are currently used, the average points score (APS) at Key Stage 2 and value-added (VA), which measures the rate of academic improvement between Key Stage 1 and 2. These figures are used by parents and the Office for Standards in Education to make judgements and comparisons. However, simple…

  9. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  10. Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.

    PubMed

    Della Ducata, Daniela; Jaehrling, Jan; Hänel, Cornelia; Satzger, Marion; Wolber, Meike; Ostendorp, Ralf; Pabst, Stefan; Brocks, Bodo

    2015-12-01

    The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.

  11. Novel Kac-Moody-type affine extensions of non-crystallographic Coxeter groups

    NASA Astrophysics Data System (ADS)

    Dechant, Pierre-Philippe; Bœhm, Céline; Twarock, Reidun

    2012-07-01

    Motivated by recent results in mathematical virology, we present novel asymmetric {Z}[\\tau ]-integer-valued affine extensions of the non-crystallographic Coxeter groups H2, H3 and H4 derived in a Kac-Moody-type formalism. In particular, we show that the affine reflection planes which extend the Coxeter group H3 generate (twist) translations along two-, three- and five-fold axes of icosahedral symmetry, and we classify these translations in terms of the Fibonacci recursion relation applied to different start values. We thus provide an explanation of previous results concerning affine extensions of icosahedral symmetry in a Coxeter group context, and extend this analysis to the case of the non-crystallographic Coxeter groups H2 and H4. These results will enable new applications of group theory in physics (quasicrystals), biology (viruses) and chemistry (fullerenes).

  12. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation. PMID:27367467

  13. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.

  14. Two measured completely different electron affinities for atomic Eu?

    NASA Astrophysics Data System (ADS)

    Msezane, A. Z.; Felfli, Z.

    2016-05-01

    Recently, the electron affinity (EA) of atomic Eu was measured to be 0.116?eV. This value is in outstanding agreement with the theoretically calculated values using the Regge pole and MCDF-RCI methods. Previously, the EA of Eu was measured to be 1.053 eV. In an attempt to resolve the discrepancy between the two measured values, we have adopted the complex angular momentum (CAM) method and investigated in the electron energy range 0.11 eV value of 2.63 eV as the EA of Eu. This leads us to conclude that neither the claimed measured EA of Eu correspond to the actual EA of Eu. We conclude that the EA in corresponds to the BE of an excited (metastable) state of the Euanion and that in to a shape resonance. We have also investigated the EA of atomic Nd and found the value of 1.88 eV, consistent with the measurement. These significant EA values of Eu and Nd could be important in the use of their negative ions in catalyzing the oxidation of water to peroxide and of methane to methanol without CO2 emission. These new results call for immediate experimental and theoretical verification.

  15. WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid): a novel, EAAT2-preferring, nonsubstrate inhibitor of high-affinity glutamate uptake

    PubMed Central

    Dunlop, John; Eliasof, Scott; Stack, Gary; McIlvain, H Beal; Greenfield, Alexander; Kowal, Dianne; Petroski, Robert; Carrick, Tikva

    2003-01-01

    The pharmacological profile of a novel glutamate transport inhibitor, WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY-855 inhibited glutamate uptake mediated by all three subtypes in a concentration-dependent manner, with preferential inhibition of the CNS-predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 μM, respectively, while EAAT1 activity was inhibited by 50% at 100 μM (IC50 values determined in oocytes were 1.3 μM (EAAT2), 52.5 μM (EAAT3) and 125.9 μM (EAAT1)). Application of WAY-855 to EAAT-expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]D-aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY-855 inhibited D-aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY-855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY-855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes. PMID:14517179

  16. Smooth big bounce from affine quantization

    NASA Astrophysics Data System (ADS)

    Bergeron, Hervé; Dapor, Andrea; Gazeau, Jean Pierre; Małkiewicz, Przemysław

    2014-04-01

    We examine the possibility of dealing with gravitational singularities on a quantum level through the use of coherent state or wavelet quantization instead of canonical quantization. We consider the Robertson-Walker metric coupled to a perfect fluid. It is the simplest model of a gravitational collapse, and the results obtained here may serve as a useful starting point for more complex investigations in the future. We follow a quantization procedure based on affine coherent states or wavelets built from the unitary irreducible representation of the affine group of the real line with positive dilation. The main issue of our approach is the appearance of a quantum centrifugal potential allowing for regularization of the singularity, essential self-adjointness of the Hamiltonian, and unambiguous quantum dynamical evolution.

  17. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  18. Artificial Affinity Proteins as Ligands of Immunoglobulins

    PubMed Central

    Mouratou, Barbara; Béhar, Ghislaine; Pecorari, Frédéric

    2015-01-01

    A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized. PMID:25647098

  19. Permeability of self-affine rough fractures

    PubMed

    Drazer; Koplik

    2000-12-01

    The permeability of two-dimensional fractures with self-affine fractal roughness is studied via analytic arguments and numerical simulations. The limit where the roughness amplitude is small compared with average fracture aperture is analyzed by a perturbation method, while in the opposite case of narrow aperture, we use heuristic arguments based on lubrication theory. Numerical simulations, using the lattice Boltzmann method, are used to examine the complete range of aperture sizes, and confirm the analytic arguments. PMID:11138092

  20. Theoretical study for the electron affinities of negative ions with the MCDHF method

    NASA Astrophysics Data System (ADS)

    Li, Junqin; Zhao, Zilong; Andersson, Martin; Zhang, Xuemei; Chen, Chongyang

    2012-08-01

    Systematic theoretical calculations based on the multi-configuration Dirac-Hartree-Fock method have been carried out for the electron affinities of anions of the elements of group III (B, Al, Ga, In and Tl), group IV (C, Si, Ge, Sn and Pb), group V (N, P and As), group VI (O, S, Se, Te and Po) and group VII (F, Cl, Br, I and At) by studying the ground energies of neutral atoms and their corresponding negative ions. The differences between the calculated total energies of the neutral atom and its anion were used to obtain the electron affinities. We discuss in detail the effects of configuration interaction, investigate the importance of including different types of correlations and check the impact of the higher order relativistic corrections on electron affinities. Our calculated electron affinities are compared with experimental and other available theoretical results. The present studies are the first systematic studies of all these elements. We give the first theoretical values for the affinities of elements Se, Te, Po and At; thereinto, there is no experimental value for elements Po and At.

  1. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  2. Overview of affinity biosensors in food analysis.

    PubMed

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field.

  3. Overview of affinity biosensors in food analysis.

    PubMed

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field. PMID:16792079

  4. A MEMS Dielectric Affinity Glucose Biosensor

    PubMed Central

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-01-01

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  5. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  6. Trematode hemoglobins show exceptionally high oxygen affinity.

    PubMed

    Kiger, L; Rashid, A K; Griffon, N; Haque, M; Moens, L; Gibson, Q H; Poyart, C; Marden, M C

    1998-08-01

    Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

  7. Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

    PubMed

    Ball, D J; Nishimura, J S

    1980-11-25

    Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits. PMID:7430155

  8. Associations of Teacher Credibility and Teacher Affinity with Learning Outcomes in Health Classrooms

    ERIC Educational Resources Information Center

    Gray, DeLeon L.; Anderman, Eric M.; O'Connell, Ann A.

    2011-01-01

    In the present study (N = 633), we examine the role of teacher credibility and teacher affinity in classrooms. We explore the relations among these two characteristics and student gains in knowledge and valuing of learning about HIV and pregnancy prevention across high school classrooms. Results marshaled support for the notion that teacher…

  9. Effects of whole-body irradiation on antibody affinity.

    PubMed Central

    Gorini, G; Adorini, L; Boraschi, D; Di Michele, A; Doria, G

    1977-01-01

    Mice exposed to a sublethal dose of X-rays were immunized with alum-precipitated DNP-KLH (dinitrophenyl-keyhole limpet haemocyanin) and B. pertussis either before or after irradiation. The primary anti-DNP antibody response was evaluated during 8 weeks after immunization by the equilibrium dialysis technique using ammonium sulphate- precipitated serum globulins and the ligand 3H-labelled xi-DNP-L-Lysine. The serum concentrations of antibody sites in mice immunized 1-5 days before or 2 h-8 weeks after 450 rad were below the values in unirradiated controls at all bleeding times. Antibody affinity, however, was found to be up to 20 fold higher in irradiated mice than in control mice when antigen was injected before, or 3-8 weeks after, irradiation. Spleen cells from mice exposed to 450 rad 1-9 weeks before killing were stimulated in vitro with PHA, ConA, or LPS. Recovery profiles of mitotic responsiveness suggest that enhancement of antibody affinity in irradiated mice could result from relative lack of suppressor T Cells. PMID:198358

  10. Boronate affinity adsorption of RNA: possible role of conformational changes

    NASA Technical Reports Server (NTRS)

    Singh, N.; Willson, R. C.; Fox, G. E. (Principal Investigator)

    1999-01-01

    Batch equilibrium adsorption isotherm determination is used to characterize the adsorption of mixed yeast RNA on agarose-immobilized m-aminophenylboronic acid. It is shown that the affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium. This adsorption-promoting influence of barium is suggested to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected. The substitution of barium for the standard magnesium counterion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations. RNA adsorption isotherms exhibit sharp transitions as functions of temperature, and these transitions occur at different temperatures with Mg2+ and with Ba2+. Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process. The stoichiometric displacement parameter, Z, in Ba2+ buffer is three times the value in Mg2+ buffer, and is close to unity.

  11. Characterization of methacrylate chromatographic monoliths bearing affinity ligands.

    PubMed

    Černigoj, Urh; Vidic, Urška; Nemec, Blaž; Gašperšič, Jernej; Vidič, Jana; Lendero Krajnc, Nika; Štrancar, Aleš; Podgornik, Aleš

    2016-09-16

    We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher. PMID:27554023

  12. Calculation of the Ionization Potentials and Electron Affinities for Atoms

    NASA Astrophysics Data System (ADS)

    Chen, Jiqiang; Krieger, J. B.; Iafrate, G. J.; Savin, A.

    1998-03-01

    The method employing the self-interaction-corrected correlation energy functional obtained from the homogeneous electron gas with a gap is extended to atoms and ions with non-zero spin polarization. As in the case for atoms and ions with ζ=3D0, the error in the calculated Ec is significantly smaller than in the LSD approximation with zero gap for atoms and ions with Z<=18. Comparison of the resulting ionization potentials and electron affinities with experimental values will also be presented. Finally, we will discuss the possibility of obtaining saturation for Ec for the He, Li, N, O, F and Ne isoelectronic series, but a divergent Ec for the Be, B and C isoelectronic series, in the large Z limit.

  13. Class II-restricted T cell receptor engineered in vitro for higher affinity retains peptide specificity and function

    PubMed Central

    Weber, K. Scott; Donermeyer, David L.; Allen, Paul M.; Kranz, David M.

    2005-01-01

    The T cell receptor (TCR) αβ heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin (Hb/I-Ek), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (Vβ-linker-Vα) was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with Hb/I-Ek to isolate a panel of affinity mutants with KD values as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation. PMID:16365315

  14. Latest European coelacanth shows Gondwanan affinities.

    PubMed

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan

    2005-06-22

    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  15. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  16. Measuring an antibody affinity distribution molecule by molecule.

    PubMed

    Temirov, Jamshid P; Bradbury, Andrew R M; Werner, James H

    2008-11-15

    Single molecule fluorescence microscopy was used to observe the binding and unbinding of hapten decorated quantum dots to individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  17. Measuring an antibody affinity distribution molecule by molecule

    SciTech Connect

    Bradbury, Andrew M; Werner, James H; Temirov, Jamshid

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  18. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  19. [Affine transformation-based automatic registration for peripheral digital subtraction angiography (DSA)].

    PubMed

    Kong, Gang; Dai, Dao-Qing; Zou, Lu-Min

    2008-07-01

    In order to remove the artifacts of peripheral digital subtraction angiography (DSA), an affine transformation-based automatic image registration algorithm is introduced here. The whole process is described as follows: First, rectangle feature templates are constructed with their centers of the extracted Harris corners in the mask, and motion vectors of the central feature points are estimated using template matching technology with the similarity measure of maximum histogram energy. And then the optimal parameters of the affine transformation are calculated with the matrix singular value decomposition (SVD) method. Finally, bilinear intensity interpolation is taken to the mask according to the specific affine transformation. More than 30 peripheral DSA registrations are performed with the presented algorithm, and as the result, moving artifacts of the images are removed with sub-pixel precision, and the time consumption is less enough to satisfy the clinical requirements. Experimental results show the efficiency and robustness of the algorithm.

  20. Work function and electron affinity of the fluorine-terminated (100) diamond surface

    NASA Astrophysics Data System (ADS)

    Rietwyk, K. J.; Wong, S. L.; Cao, L.; O'Donnell, K. M.; Ley, L.; Wee, A. T. S.; Pakes, C. I.

    2013-03-01

    The work function and electron affinity of fluorine-terminated (100) diamond surfaces prepared by exposure to dissociated XeF2 have been determined using synchrotron-based photoemission. After vacuum annealing to 350 °C a clean, monofluoride terminated C(100):F surface was obtained for which an electron affinity of 2.56 eV was measured. This is the highest electron affinity reported for any diamond surface termination so far, and it exceeds the value predicted by recent density functional theory calculations by 0.43 eV. The work function of 7.24 eV measured for the same surface places the Fermi energy of 0.79 eV above the valence band maximum.

  1. Soybean. beta. -glucan binding sites display maximal affinity for a heptaglucoside phytoalexin-elicitor

    SciTech Connect

    Cosio, E.G.; Waldmueller, T.; Frey, T.; Ebel, J. )

    1990-05-01

    The affinity of soybean {beta}-glucan-binding sites for a synthetic heptaglucan elicitor was tested in a ligand-competition assay against a {sup 125}I-labeled 1,3-1,6-{beta}-glucan preparation (avg. DP=20). Half-maximal displacement of label (IC{sub 50}) was obtained at 9nM heptaglucan, the highest affinity of all fractions tested to date. Displacement followed a uniform sigmoidal pattern and was complete at 1{mu}M indicating access of heptaglucan to all sites available to the labeled elicitor. A mathematical model was used to predict IC{sub 50} values according to the DP of glucan fragments obtained from fungal cell walls. The lowest IC{sub 50} predicted by this model is 3nM. Binding affinity of the glucans was compared with their elicitor activity in a bioassay.

  2. Optimized Structures and Proton Affinities of Fluorinated Dimethyl Ethers: An Ab Initio Study

    NASA Technical Reports Server (NTRS)

    Orgel, Victoria B.; Ball, David W.; Zehe, Michael J.

    1996-01-01

    Ab initio methods have been used to investigate the proton affinity and the geometry changes upon protonation for the molecules (CH3)2O, (CH2F)2O, (CHF2)2O, and (CF3)2O. Geometry optimizations were performed at the MP2/3-2 I G level, and the resulting geometries were used for single-point energy MP2/6-31G calculations. The proton affinity calculated for (CH3)2O was 7 Kjoule/mole from the experimental value, within the desired variance of +/- 8Kjoule/mole for G2 theory, suggesting that the methodology used in this study is adequate for energy difference considerations. For (CF3)20, the calculated proton affinity of 602 Kjoule/mole suggests that perfluorinated ether molecules do not act as Lewis bases under normal circumstances; e.g. degradation of commercial lubricants in tribological applications.

  3. Avoiding degenerate coframes in an affine gauge approach to quantum gravity

    SciTech Connect

    Mielke, E.W.; McCrea, J.D.; Ne`eman, Y.; Hehl, F.W.

    1993-04-01

    This report discusses the following concepts on quantum gravity: The affine gauge approach; affine gauge transformations versus active differomorphisms; affine gauge approach to quantum gravity with topology change.

  4. Proton and sodium cation affinities of harpagide: a computational study.

    PubMed

    Colas, Cyril; Bouchonnet, Stéphane; Rogalewicz-Gilard, Françoise; Popot, Marie-Agnès; Ohanessian, Gilles

    2006-06-15

    The aim of this work was to estimate the proton and sodium cation affinities of harpagide (Har), an iridoid glycoside responsible for the antiinflammatory properties of the medicinal plant Harpagophytum. Monte Carlo conformational searches were performed at the semiempirical AM1 level to determine the most stable conformers for harpagide and its protonated and Na+-cationized forms. The 10 oxygen atoms of the molecule were considered as possible protonation and cationization sites. Geometry optimizations were then refined at the DFT B3LYP/6-31G level from the geometries of the most stable conformers found. Final energetics were obtained at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G level. The proton and sodium ion affinities of harpagide have been estimated at 223.5 and 66.0 kcal/mol, respectively. Since harpagide mainly provides HarNa+ ions in electrospray experiments, the DeltarG298 associated with the reaction of proton/sodium exchange between Har and methanol, MeOHNa+ + HarH+ --> MeOH2+ + HarNa+ (1), has been calculated; it has been estimated to be 1.9 kcal/mol. Complexing a methanol molecule to each reagent and product of reaction 1 makes the reaction become exothermic by 1.7 kcal/mol. These values are in the limit of the accuracy of the method and do not allow us to conclude definitely whether the reaction is endo- or exothermic, but, according to these very small values, the cation exchange reaction is expected to proceed easily in the final stages of the ion desolvation process. PMID:16759142

  5. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. PMID:27018856

  6. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    PubMed

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  7. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  8. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc.

  9. Kinetic and in silico studies of novel hydroxy-based thymol analogues as inhibitors of mushroom tyrosinase.

    PubMed

    Ashraf, Zaman; Rafiq, Muhammad; Seo, Sung-Yum; Kwon, Kang Sung; Babar, Mustafeez Mujtaba; Zaidi, Najam-us-Sahar Sadaf

    2015-06-15

    The present studies reports the synthesis of hydoxylated thymol analogues (4a-e) and (6a-c) as mushroom tyrosinase inhibitors. The title compounds were obtained in good yield and characterized by FTIR, (1)H NMR, (13)C NMR, Mass spectral data and X-ray crystallography in case of compound (6a). The inhibitory effects on mushroom tyrosinase and DPPH were evaluated and it was observed that 2-[5-methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate (6b) showed tyrosinase inhibitory activity (IC50 15.20 μM) comparable to kojic acid (IC50 16.69 μM) while 2-[5-methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3,4-dihydroxybenzoate (4d) exhibited higher antioxidant potential (IC50 11.30 μM) than standard ascorbic acid (IC50 24.20 μM). The docking studies of synthesized thymol analogues was also performed against tyrosinase protein (PDBID 2ZMX) to compare the binding affinities with IC50 values. The predicted binding affinities are in good agreement with the IC50 values as compound (6b) showed highest binding affinity -7.1 kcal/mol. The kinetic mechanism analyzed by Lineweaver-Burk plots exhibited that compound (4d) and (6b) inhibit the enzyme by two different pathways displayed mixed-type inhibition. The inhibition constants Ki calculated from Dixon plots for compounds (4d) and (6b) are 34 μM and 25 μM respectively. It was also found from kinetic analysis that derivative (6b) formed reversible enzyme inhibitor complex. It is propose on the basis of our investigation that title compound (6b) may serve as lead structure for the design of more potent tyrosinase inhibitors.

  10. Kinetic and in silico studies of novel hydroxy-based thymol analogues as inhibitors of mushroom tyrosinase.

    PubMed

    Ashraf, Zaman; Rafiq, Muhammad; Seo, Sung-Yum; Kwon, Kang Sung; Babar, Mustafeez Mujtaba; Zaidi, Najam-us-Sahar Sadaf

    2015-06-15

    The present studies reports the synthesis of hydoxylated thymol analogues (4a-e) and (6a-c) as mushroom tyrosinase inhibitors. The title compounds were obtained in good yield and characterized by FTIR, (1)H NMR, (13)C NMR, Mass spectral data and X-ray crystallography in case of compound (6a). The inhibitory effects on mushroom tyrosinase and DPPH were evaluated and it was observed that 2-[5-methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate (6b) showed tyrosinase inhibitory activity (IC50 15.20 μM) comparable to kojic acid (IC50 16.69 μM) while 2-[5-methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3,4-dihydroxybenzoate (4d) exhibited higher antioxidant potential (IC50 11.30 μM) than standard ascorbic acid (IC50 24.20 μM). The docking studies of synthesized thymol analogues was also performed against tyrosinase protein (PDBID 2ZMX) to compare the binding affinities with IC50 values. The predicted binding affinities are in good agreement with the IC50 values as compound (6b) showed highest binding affinity -7.1 kcal/mol. The kinetic mechanism analyzed by Lineweaver-Burk plots exhibited that compound (4d) and (6b) inhibit the enzyme by two different pathways displayed mixed-type inhibition. The inhibition constants Ki calculated from Dixon plots for compounds (4d) and (6b) are 34 μM and 25 μM respectively. It was also found from kinetic analysis that derivative (6b) formed reversible enzyme inhibitor complex. It is propose on the basis of our investigation that title compound (6b) may serve as lead structure for the design of more potent tyrosinase inhibitors. PMID:26025140

  11. Electron Affinities, Fluoride Affinities, and Heats of Formation of the Second Row Transition Metal Hexafluorides: MF6 (M = Mo, Tc, Ru, Rh, Pd, Ag)

    SciTech Connect

    Craciun, Raluca; Long, Rebecca T.; Dixon, David A.; Christe, Karl O.

    2010-07-22

    High-level electronic structure calculations were used to evaluate reliable, self-consistent thermochemical data sets for the second row transition metal hexafluorides. The electron affinities, heats of formation, first (MF{sub 6} {yields} MF{sub 5} + F) and average M-F bond dissociation energies, and fluoride affinities of MF{sub 6} (MF{sub 6} + F{sup -} {yields} MF{sub 7}{sup -}) and MF{sub 5} (MF{sub 5} + F{sup -} {yields} MF{sub 6}{sup -}) were calculated. The electron affinities are higher than those of the corresponding third row hexafluorides, making them stronger one-electron oxidizers. The calculated electron affinities, in good agreement with the available experimental values, are 4.23 eV for MoF{sub 6}, 5.89 eV for TcF{sub 6}, 7.01 eV for RuF{sub 6}, 6.80 eV for RhF{sub 6}, 7.95 eV for PdF{sub 6}, and 8.89 eV for AgF{sub 6}. The corresponding pentafluorides are also very strong Lewis acids, although their acidities on the pF{sup -} scale are about one unit lower than those of the third row pentafluorides. The performance of a wide range of DFT exchange-correlation functionals was benchmarked by comparing them to our more accurate CCSD(T) results.

  12. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  13. High-affinity Cyclic Peptide Matriptase Inhibitors*

    PubMed Central

    Quimbar, Pedro; Malik, Uru; Sommerhoff, Christian P.; Kaas, Quentin; Chan, Lai Y.; Huang, Yen-Hua; Grundhuber, Maresa; Dunse, Kerry; Craik, David J.; Anderson, Marilyn A.; Daly, Norelle L.

    2013-01-01

    The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase. PMID:23548907

  14. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  15. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  16. Determination of proton affinities and acidity constants of sugars.

    PubMed

    Feng, Shuting; Bagia, Christina; Mpourmpakis, Giannis

    2013-06-20

    Proton transfer reactions play a key role in the conversion of biomass derived sugars to chemicals. In this study, we employ high level ab initio theoretical methods, in tandem with solvation effects to calculate the proton affinities (PA) and acidity constants (pKa) of various d-glucose and d-fructose tautomers (protonation-deprotonation processes). In addition, we compare the theoretically derived pH values of sugar solutions against experimentally measured pH values in our lab. Our results demonstrate that the protonation of any of the O atoms of the sugars is thermodynamically preferred without any significant variation in the PA values. Intramolecular hydrogen transfers, dehydration reactions, and ring-opening processes were observed, resulting from the protonation of specific hydroxyl groups on the sugars. Regarding the deprotonation processes (pKa), we found that the sugars' anomeric hydroxyls exhibit the highest acidity. The theoretically calculated pH values of sugar solutions are in excellent agreement with experimental pH measurements at low sugar concentrations. At higher sugar concentrations the calculations predict less acidic solutions than the experiments. In this case, we expect the sugars to act as solvents increasing the proton solvation energy and the acidity of the solutions. We demonstrated through linear relationships that the pKa values are correlated with the relative stability of the conjugate bases. The latter is related to hydrogen bonding and polarization of the C-O(-) bond. A plausible explanation for the good performance of the direct method in calculating the pKa values of sugars can be the presence of intramolecular hydrogen bonds on the conjugate base. Both theory and experiments manifest that fructose is a stronger acid than glucose, which is of significant importance in self-catalyzed biomass-relevant dehydration reactions. PMID:23706015

  17. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  18. Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity.

    PubMed Central

    Gennis, L S

    1976-01-01

    A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+.Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested. PMID:186779

  19. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

    PubMed Central

    Day, Christopher J.; Tran, Elizabeth N.; Semchenko, Evgeny A.; Tram, Greg; Hartley-Tassell, Lauren E.; Ng, Preston S. K.; King, Rebecca M.; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A.; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P.

    2015-01-01

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein–glycan or protein–protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host–glycan:bacterial–glycan pairs with equilibrium dissociation constants (KD) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  20. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

    PubMed

    Day, Christopher J; Tran, Elizabeth N; Semchenko, Evgeny A; Tram, Greg; Hartley-Tassell, Lauren E; Ng, Preston S K; King, Rebecca M; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P

    2015-12-29

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  1. Phosphatidylserine Reversibly Binds Cu2+ with Extremely High Affinity

    PubMed Central

    Monson, Christopher F.; Cong, Xiao; Robison, Aaron; Pace, Hudson P.; Liu, Chunming; Poyton, Matthew F.; Cremer, Paul S.

    2012-01-01

    Phosphatidylserine (PS) embedded within supported lipid bilayers (SLBs) was found to bind Cu2+ from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu2+ to PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85–90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu2+ concentrations and basic values pH (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion limited complex formation at basic pH, but rapid dissociation under acidic conditions. The tight binding of Cu2+ to PS may have physiological consequences under certain circumstances. PMID:22548290

  2. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  3. Effectively nonlocal metric-affine gravity

    NASA Astrophysics Data System (ADS)

    Golovnev, Alexey; Koivisto, Tomi; Sandstad, Marit

    2016-03-01

    In metric-affine theories of gravity such as the C-theories, the spacetime connection is associated to a metric that is nontrivially related to the physical metric. In this article, such theories are rewritten in terms of a single metric, and it is shown that they can be recast as effectively nonlocal gravity. With some assumptions, known ghost-free theories with nonsingular and cosmologically interesting properties may be recovered. Relations between different formulations are analyzed at both perturbative and nonperturbative levels, taking carefully into account subtleties with boundary conditions in the presence of integral operators in the action, and equivalences between theories related by nonlocal redefinitions of the fields are verified at the level of equations of motion. This suggests a possible geometrical interpretation of nonlocal gravity as an emergent property of non-Riemannian spacetime structure.

  4. Affinity chromatography with an immobilized RNA enzyme.

    PubMed Central

    Vioque, A; Altman, S

    1986-01-01

    M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid. Images PMID:3526344

  5. Wetting on rough self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Palasantzas, George

    1995-05-01

    In this paper, we present a general investigation of the effective potential for complete wetting on self-affine rough surfaces. The roughness effect is investigated by means of the height-height correlation model in Fourier space ~(1+aξ2q2)-1-H. The parameters H and ξ are, respectively, the roughness exponent and the substrate in-plane correlation length. It is observed that the effect of H on the free interface profile is significant for ξ>ξ) regime is characterized by a power-law scaling ~Y-2.

  6. High-affinity neuropeptide Y receptor antagonists.

    PubMed Central

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J

    1995-01-01

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats. PMID:7568074

  7. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field. PMID:25570332

  8. Evaluation system of negative electron affinity photocathode

    NASA Astrophysics Data System (ADS)

    Fu, Rongguo; Chang, Benkang; Qian, Yunsheng; Wang, Guihua; Zong, Zhiyuan

    2001-10-01

    This article first describes the background of the research and manufacture of evaluation system of Negative Electron Affinity photocathode. This article designs a set of super high vacuum system for activating NEA photocathode on the base of activation theory, the process of design and debugging is given. The system is composed of three parts: super high vacuum system for GaAs material activation, multi-meter testing system, surface analysis system. The system is used for on-line evaluation of activating of NEA photocathode. The technical parameters and structure of the evaluation system of NEA photocathode are given in the paper. The system is finished and experiments are made. At last the picture of the system is given.

  9. Identification of high-affinity anti-IL-1. alpha. autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1. alpha

    SciTech Connect

    Mae, N.; Liberato, D.J.; Chizzonite, R.; Satoh, H. )

    1991-04-01

    A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 {alpha} (rhIL-1 alpha) has been developed. Results from this ELISA have demonstrated that the concentration of rhIL-1 {alpha} added to normal human serum (NHS) decreased by 16.3% after 3 h and 24.9% after 6 h at room temperature. Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 {alpha} added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation. The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 {alpha} in NHS. Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 {alpha} binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 {alpha}-specific IgG. The binding of 125I-labeled IL-1 {alpha} to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 7.4 {times} 10(-11) M) but not by unlabeled IL-1 {beta}. Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 {alpha}-specific IgGs was 4.7 {times} 10(10) M-1. These results suggest that these autoantibodies may interfere with the detection of IL-1 {alpha} in human serum by various assay systems and also could be a regulator of circulating IL-1 {alpha}.

  10. Purification of glycolytic enzymes by using affinity-elution chromatography.

    PubMed Central

    Scopes, R K

    1977-01-01

    1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures. PMID:192194

  11. Antibody-based affinity cryo-EM grid.

    PubMed

    Yu, Guimei; Li, Kunpeng; Jiang, Wen

    2016-05-01

    The Affinity Grid technique combines sample purification and cryo-Electron Microscopy (cryo-EM) grid preparation into a single step. Several types of affinity surfaces, including functionalized lipids monolayers, streptavidin 2D crystals, and covalently functionalized carbon surfaces have been reported. More recently, we presented a new affinity cryo-EM approach, cryo-SPIEM, which applies the traditional Solid Phase Immune Electron Microscopy (SPIEM) technique to cryo-EM. This approach significantly simplifies the preparation of affinity grids and directly works with native macromolecular complexes without need of target modifications. With wide availability of high affinity and high specificity antibodies, the antibody-based affinity grid would enable cryo-EM studies of the native samples directly from cell cultures, targets of low abundance, and unstable or short-lived intermediate states.

  12. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  13. Concurrent low- and high-affinity sulfate reduction kinetics in marine sediment

    NASA Astrophysics Data System (ADS)

    Harder Tarpgaard, Irene; Røy, Hans; Jørgensen, Bo Barker

    Bacterial sulfate reduction in marine sediments generally occurs in the presence of high millimolar concentrations of sulfate. Published data indicate that low sulfate concentrations may limit sulfate reduction rates below 0.2-2 mM. Yet, high sulfate reduction rates occur in the 1-100 μM range in freshwater sediments and at the sulfate-methane transition in marine sediments. Through a combination of 35S-tracer experiments, including initial velocity experiments and time course experiments, we searched for different sulfate affinities in the mixed community of sulfate reducers in a marine sediment. We supported the radiotracer experiments with a highly sensitive ion chromatographic technique for sulfate with a detection limit of 0.15 μM SO 42- in marine pore water. Our results showed that high and low affinities for sulfate co-occur and that the applied experimental approach may determine the observed apparent half saturation constant, Km. Our experimental and model data both show that sulfate reduction in the studied marine sediment could be explained by two dominating affinities for sulfate: a low affinity with a mean half saturation constant, Km, of 430 μM SO 42- and a high affinity with a mean Km of 2.6 μM SO 42-. The high-affinity sulfate reduction was thermodynamically un-constrained down to <1 μM SO 42-, both in our experiments and under in situ conditions. The reduction of radio-labeled sulfate was partly reversible due to concurrent re-oxidation of sulfide by Fe(III) and possibly due to a reversibility of the enzymatic pathway of sulfate reduction. A literature survey of apparent Km values for sediments and pure cultures is presented and discussed.

  14. Kinetics and molecular docking studies of the inhibitions of angiotensin converting enzyme and renin activities by hemp seed (Cannabis sativa L.) peptides.

    PubMed

    Girgih, Abraham T; He, Rong; Aluko, Rotimi E

    2014-05-01

    Four novel peptide sequences (WVYY, WYT, SVYT, and IPAGV) identified from an enzymatic digest of hemp seed proteins were used for enzyme inhibition kinetics and molecular docking studies. Results showed that WVYY (IC50 = 0.027 mM) was a more potent (p < 0.05) ACE-inhibitory peptide than WYT (IC50 = 0.574 mM). However, WYT (IC50 = 0.054 mM) and SVYT (IC50 = 0.063 mM) had similar renin-inhibitory activity, which was significantly better than that of IPAGV (IC50 = 0.093 mM). Kinetics studies showed that WVYY had a lower inhibition constant (Ki) of 0.06 mM and hence greater affinity for ACE when compared to the 1.83 mM obtained for WYT. SVYT had lowest Ki value of 0.89 mM against renin, when compared to the values obtained for WYT and IPAGV. Molecular docking results showed that the higher inhibitory activities of WVYY and SVYT were due to the greater degree of noncovalent bond-based interactions with the enzyme protein, especially formation of higher numbers of hydrogen bonds with active site residues.

  15. Affinity chromatography for purification of two urokinases from human urine.

    PubMed

    Takahashi, R; Akiba, K; Koike, M; Noguchi, T; Ezure, Y

    2000-05-26

    A new affinity chromatography (hydrophobic-mediated affinity chromatography), which was characterized by the matrix having both affinity site to urokinase and hydrophobic site, was established for the purification of urokinase from human urine. The hydrophobic affinity matrix (tentatively named PAS in the text) was prepared by immobilizing 6-aminocaproic acid on Sepharose CL-6B, followed by a coupling p-aminobenzamidine to a part of the hydrophobic site on the matrix. The PAS matrix was applied to the purification of urokinase from human urine, and high- and low-molecular weight pure urokinases were efficiently obtained in high yield by the present method. PMID:10892585

  16. Complex Affine Toda Theories and Soliton Solutions

    NASA Astrophysics Data System (ADS)

    Zhu, Zhiqing

    1995-01-01

    Toda field theories (TFT's) constitute a large class of integrable (1 + 1)-dimensional field theories that are relativistically invariant: included are conformal field theories and integrable deformations away from conformality. Because they are soluble, for example, by the inverse scattering method, and because they are related to many other areas of field theory, they have been studied extensively in recent years. Hirota's method is a straightforward procedure to obtain soliton solutions to non-linear integrable equations. In Hirota's method, one first writes the nonlinear equations in Hirota's bilinear form, and then expands the so called tau-functions as a power series in an arbitrary parameter. The power series terminates at some finite order, thus the solutions obtained are exact. For an N-soliton solution, the number of terms in the expansion grows exponentially with N, making direct calculation of N-soliton solutions difficult. We extend Hirota's one -parameter expansion to an N-parameter expansion. In the new expansion series, many terms are identical to those in the (N - 1)-soliton solutions, and new terms grow only linearly with N. Furthermore, we note that the expansion must terminate at some finite order, thus the vanishing of higher order terms can be used as constraints on these new terms. It turns out that these constraints can be used to determine the new terms completely. We used this extended Hirota's method to find N-soliton solutions for complex affine TFT's based on a simply-laced Kac-Moody algebra. Soliton solutions for non-simply-laced complex ATFT's can be obtained for those of simply-laced complex ATFT's by folding or twisting. Even though some soliton solutions have already been obtained for complex ATFT's by various methods, the physical implications of these solutions have not yet been thoroughly discussed. There are infinitely many distinct topological solitons in any given complex affine Toda field theory and most of them have complex

  17. Associations of teacher credibility and teacher affinity with learning outcomes in health classrooms

    PubMed Central

    Anderman, Eric M.; O’Connell, Ann A.

    2011-01-01

    In the present study (N = 633), we examine the role of teacher credibility and teacher affinity in classrooms. We explore the relations among these two characteristics and student gains in knowledge and valuing of learning about HIV and pregnancy prevention across high school classrooms. Results marshaled support for the notion that teacher characteristics are associated with classroom-level gains in learning outcomes. Above and beyond student-level predictors, teacher credibility (aggregated to the classroom level) was positively related to increases in knowledge across classrooms, whereas aggregated teacher affinity was positively related to an increased valuing of learning about HIV and pregnancy prevention across classrooms. Future directions and implications for practice are discussed. PMID:24876800

  18. Determination of electron affinity of carbonyl radicals by means of negative ion mass spectrometry.

    PubMed

    Muftakhov; Vasil'ev; Mazunov

    1999-06-01

    Appearance energies of [M-H](-) ions from carbonyl compounds R-CO-R' (R,R' = H, CH(3), NH(2), OH) have been measured by means of negative ion mass spectrometry in resonant electron capture mode. Values of electron affinity of the corresponding radicals, CH(2)&dbond;C(X)O, NH&dbond;C(X)O and O&dbond;C(X)O, have been determined. Copyright 1999 John Wiley & Sons, Ltd. PMID:10407285

  19. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  20. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  1. Ligand Affinities Estimated by Quantum Chemical Calculations.

    PubMed

    Söderhjelm, Pär; Kongsted, Jacob; Ryde, Ulf

    2010-05-11

    We present quantum chemical estimates of ligand-binding affinities performed, for the first time, at a level of theory for which there is a hope that dispersion and polarization effects are properly accounted for (MP2/cc-pVTZ) and at the same time effects of solvation, entropy, and sampling are included. We have studied the binding of seven biotin analogues to the avidin tetramer. The calculations have been performed by the recently developed PMISP approach (polarizable multipole interactions with supermolecular pairs), which treats electrostatic interactions by multipoles up to quadrupoles, induction by anisotropic polarizabilities, and nonclassical interactions (dispersion, exchange repulsion, etc.) by explicit quantum chemical calculations, using a fragmentation approach, except for long-range interactions that are treated by standard molecular-mechanics Lennard-Jones terms. In order to include effects of sampling, 10 snapshots from a molecular dynamics simulation are studied for each biotin analogue. Solvation energies are estimated by the polarized continuum model (PCM), coupled to the multipole-polarizability model. Entropy effects are estimated from vibrational frequencies, calculated at the molecular mechanics level. We encounter several problems, not previously discussed, illustrating that we are first to apply such a method. For example, the PCM model is, in the present implementation, questionable for large molecules, owing to the use of a surface definition that gives numerous small cavities in a protein. PMID:26615702

  2. Affinity of guanosine derivatives for polycytidylate revisited

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Hurley, T. B.; Baird, E. E.

    1995-01-01

    Evidence is presented for complexation of guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23 degrees C in the presence of 1.0 M NaCl2 and 0.2 M MgCl2 in water. The association of 2-MeImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) 2-MeImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-MeImpG equal to 5.55 +/- 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5'-monophosphate (5'GMP), guanosine 5'-monophosphate imidazolide (ImpG), and guanosine 5'-monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-MeImpG.

  3. Prostate Cancer and Bone: The Elective Affinities

    PubMed Central

    2014-01-01

    The onset of metastases dramatically changes the prognosis of prostate cancer patients, determining increased morbidity and a drastic fall in survival expectancy. Bone is a common site of metastases in few types of cancer, and it represents the most frequent metastatic site in prostate cancer. Of note, the prevalence of tumor relapse to the bone appears to be increasing over the years, likely due to a longer overall survival of prostate cancer patients. Bone tropism represents an intriguing challenge for researchers also because the preference of prostate cancer cells for the bone is the result of a sequential series of targetable molecular events. Many factors have been associated with the peculiar ability of prostate cancer cells to migrate in bone marrow and to determine mixed osteoblastic/osteolytic lesions. As anticipated by the success of current targeted therapy aimed to block bone resorption, a better understanding of molecular affinity between prostate cancer and bone microenvironment will permit us to cure bone metastasis and to improve prognosis of prostate cancer patients. PMID:24971315

  4. Banach frames in the affine synthesis problem

    NASA Astrophysics Data System (ADS)

    Terekhin, Pavel A.

    2009-10-01

    We consider the problem of representing functions f\\in L^p(\\mathbb R^d) by a series in elements of the affine system \\displaystyle \\psi_{j,k}(x)=\\lvert\\det a_j\\rvert^{1/2}\\psi(a_jx-bk), \\qquad j\\in\\mathbb N, \\quad k\\in\\mathbb Z^d. The corresponding representation theorems are established on the basis of the frame inequalities \\displaystyle A\\Vert g\\Vert _q\\le\\Vert\\{(g,\\psi_{j,k})\\}\\Vert _Y\\le B\\Vert g\\Vert _q for the Fourier coefficients \\displaystyle(g,\\psi_{j,k})=\\int_{\\mathbb R^d}g(x)\\psi_{j,k}(x)\\,dx of functions g\\in L^q(\\mathbb R^d), 1/p+1/q=1, where {\\Vert\\cdot\\Vert}_Y is the norm in some Banach space of number families \\{y_{j,k}\\} and 0 are constants. In particular, it is proved that if the integral of a function \\psi\\in L^1\\cap L^p(\\mathbb R^d), 1, is nonzero, so \\displaystyle\\int_{\\mathbb R^d}\\psi(x)\\,dx\

  5. CSAR Data Set Release 2012: Ligands, Affinities, Complexes, and Docking Decoys

    PubMed Central

    2013-01-01

    A major goal in drug design is the improvement of computational methods for docking and scoring. The Community Structure Activity Resource (CSAR) has collected several data sets from industry and added in-house data sets that may be used for this purpose (www.csardock.org). CSAR has currently obtained data from Abbott, GlaxoSmithKline, and Vertex and is working on obtaining data from several others. Combined with our in-house projects, we are providing a data set consisting of 6 protein targets, 647 compounds with biological affinities, and 82 crystal structures. Multiple congeneric series are available for several targets with a few representative crystal structures of each of the series. These series generally contain a few inactive compounds, usually not available in the literature, to provide an upper bound to the affinity range. The affinity ranges are typically 3–4 orders of magnitude per series. For our in-house projects, we have had compounds synthesized for biological testing. Affinities were measured by Thermofluor, Octet RED, and isothermal titration calorimetry for the most soluble. This allows the direct comparison of the biological affinities for those compounds, providing a measure of the variance in the experimental affinity. It appears that there can be considerable variance in the absolute value of the affinity, making the prediction of the absolute value ill-defined. However, the relative rankings within the methods are much better, and this fits with the observation that predicting relative ranking is a more tractable problem computationally. For those in-house compounds, we also have measured the following physical properties: logD, logP, thermodynamic solubility, and pKa. This data set also provides a substantial decoy set for each target consisting of diverse conformations covering the entire active site for all of the 58 CSAR-quality crystal structures. The CSAR data sets (CSAR-NRC HiQ and the 2012 release) provide substantial, publically

  6. Binding affinities and thermodynamics of noncovalent functionalization of carbon nanotubes with surfactants.

    PubMed

    Oh, Hyunkyu; Sim, Jinsook; Ju, Sang-Yong

    2013-09-01

    Binding affinity and thermodynamic understanding between a surfactant and carbon nanotube is essential to develop various carbon nanotube applications. Flavin mononucleotide-wrapped carbon nanotubes showing a large redshift in optical signature were utilized to determine the binding affinity and related thermodynamic parameters of 12 different nanotube chiralities upon exchange with other surfactants. Determined from the midpoint of sigmoidal transition, the equilibrium constant (K), which is inversely proportional to the binding affinity of the initial surfactant-carbon nanotube, provided quantitative binding strengths of surfactants as SDBS > SC ≈ FMN > SDS, irrespective of electronic types of SWNTs. Binding affinity of metallic tubes is weaker than that of semiconducting tubes. The complex K patterns from semiconducting tubes show preference to certain SWNT chiralities and surfactant-specific cooperativity according to nanotube chirality. Controlling temperature was effective to modulate K values by 30% and enables us to probe thermodynamic parameters. Equally signed enthalpy and entropy changes produce Gibbs energy changes with a magnitude of a few kJ/mol. A greater negative Gibbs energy upon exchange of surfactant produces an enhanced nanotube photoluminescence, implying the importance of understanding thermodynamics for designing nanotube separation and supramolecular assembly of surfactant.

  7. QSAR modeling of globulin binding affinity of corticosteroids using AM1 calculations.

    PubMed

    De, Kakali; Sengupta, Chandana; Roy, Kunal

    2004-06-15

    A quantitative structure-activity analysis of binding affinity of a series of 30 steroids for corticosteroid-binding globulin was performed using Wang-Ford charges of the non-hydrogen common atoms obtained from molecular electrostatic potential surface of AM1 optimized energy-minimized geometries of the compounds. Attempts were made to include lipophilicity (logP) and molar refractivity (MR) values of the whole molecules in the multivariate relations. The final relations were subjected to 'leave-one-out' cross-validation to check their predictive potential. It was found from the study that the charges of different atoms of the steroid nucleus [atoms 3, 4, 5 (ring A), 8, 9 (fusion points of rings B and C) and 16 (ring D)] contribute significantly to the binding affinity. This suggests the importance of these atoms/sites for the globulin binding affinity, which is also supported by previous reports on structure-activity relations of corticosteroids. Further, molar refractivity shows parabolic relation with the binding affinity, which indicates the possibility of dispersion interactions. The statistical qualities of the final equations generated in the present study (predicted variance 77-82%; explained variance 83-87%) are better than those of some of the previously reported models.

  8. Chemiluminescently labeled aptamers as the affinity probe for interaction analysis by capillary electrophoresis.

    PubMed

    Li, Hong-Yi; Deng, Qin-Pei; Zhang, De-Wen; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2010-07-01

    Aptamers are nucleic acid oligonucleotides, which can recognize targets with high affinity and specificity. Fluorescently labeled aptamers have been used as affinity probes in CE for interaction analysis. In this study, a method of labeling aptamers chemiluminescently with isoluminol isothiocyanate (ILITC) through covalent bonds was proposed and realized. The ILITC-labeled aptamers were characterized by HPLC-MS and purified by HPLC. After desalination, the ILITC-labeled aptamers were employed as the affinity probe for interaction analysis in CE coupled with chemiluminescence detection (CE-CL) by interface of end column reaction mode, the apparatus of which was home-designed and setup. CE-CL experiment conditions, including buffer pH, concentrations of horseradish peroxidase and H(2)O(2), were optimized first. The system of thrombin and its 29-mer aptamer was chosen as the model. Binding parameters, namely the dissociation constant (K(d)) and the binding site number (n), were calculated. The K(d) obtained was 124.0+/-6.9 nM in agreement with the reported values. Thus, interaction analysis method based on chemiluminescently labeled aptamers as the affinity probe in CE-CL has been established. This method can be widely applied due to the ease and universality of the labeling method, simplicity of CE-CL apparatus and combination with aptamers for a wide range of targets.

  9. Rationally manipulating aptamer binding affinities in a stem-loop molecular beacon.

    PubMed

    Armstrong, Rachel E; Strouse, Geoffrey F

    2014-10-15

    Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM - 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the Kd is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed Kd. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate Kd's for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes.

  10. Fluorotrimethylsilane affinities of anionic nucleophiles: a study of fluoride-induced desilylation.

    PubMed

    Krouse, Ian H; Wenthold, Paul G

    2005-05-01

    In this study, preparation and decomposition of five novel pentavalent fluorosiliconates, RSi(CH3)3F- (R = CH3CH2O, CF3CH2O, (CH3)2CHO, (CH3)3SiO, and (CH3)3SiNH) is used to investigate the process of fluoride-induced desilylation. The siliconates were characterized by collision-induced dissociation and energy-resolved mass spectrometry. Decomposition of RSi(CH3)3F- leads to loss of the nucleophile R- and FSi(CH3)3, except in the case of (CH3)3SiNHSi(CH3)3F-, where HF loss is also observed. Ion affinities for FSi(CH3)3 have been measured for all five nucleophiles, and compare well with computational predictions. The observed trend of the bond dissociation energies resembles the trend of deltaH(acid) values for the corresponding conjugate acids, RH. Additionally, this data has been incorporated with existing thermochemistry to derive fluoride affinities for four of the silanes (R = CH3CH2O, (CH3)2CHO, (CH3)3SiO, and (CH3)3SiNH). We use the fluoride affinity of the silanes and the FSi(CH3)3 affinity of the departing nucleophilic anion to assess the feasibility of fluoride-induced desilylation of the silanes examined in this work. PMID:15862771

  11. The low-affinity complex of cytochrome c and its peroxidase

    PubMed Central

    Van de Water, Karen; Sterckx, Yann G. J.; Volkov, Alexander N.

    2015-01-01

    The complex of yeast cytochrome c peroxidase and cytochrome c is a paradigm of the biological electron transfer (ET). Building on seven decades of research, two different models have been proposed to explain its functional redox activity. One postulates that the intermolecular ET occurs only in the dominant, high-affinity protein–protein orientation, while the other posits formation of an additional, low-affinity complex, which is much more active than the dominant one. Unlike the high-affinity interaction—extensively studied by X-ray crystallography and NMR spectroscopy—until now the binding of cytochrome c to the low-affinity site has not been observed directly, but inferred mainly from kinetics experiments. Here we report the structure of this elusive, weak protein complex and show that it consists of a dominant, inactive bound species and an ensemble of minor, ET-competent protein–protein orientations, which summarily account for the experimentally determined value of the ET rate constant. PMID:25944250

  12. Striving for Empathy: Affinities, Alliances and Peer Sexuality Educators

    ERIC Educational Resources Information Center

    Fields, Jessica; Copp, Martha

    2015-01-01

    Peer sexuality educators' accounts of their work reveal two approaches to empathy with their students: affinity and alliance. "Affinity-based empathy" rests on the idea that the more commonalities sexuality educators and students share (or perceive they share), the more they will be able to empathise with one another, while…

  13. Affine group formulation of the Standard Model coupled to gravity

    SciTech Connect

    Chou, Ching-Yi; Ita, Eyo; Soo, Chopin

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  14. Tending to Change: Toward a Situated Model of Affinity Spaces

    ERIC Educational Resources Information Center

    Bommarito, Dan

    2014-01-01

    The concept of affinity spaces, a theoretical construct used to analyze literate activity from a spatial perspective, has gained popularity among scholars of literacy studies and, particularly, video-game studies. This article seeks to expand current notions of affinity spaces by identifying key assumptions that have limited researchers'…

  15. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    PubMed

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  16. Steric effects on alkyl cation affinities of maingroup-element hydrides.

    PubMed

    Ruiz, Juan M; Mulder, R Joshua; Fonseca Guerra, Célia; Bickelhaupt, F Matthias

    2011-03-01

    We have carried out an extensive exploration of gas-phase alkyl cation affinities (ACA) of archetypal anionic and neutral bases across the periodic system using zeroth order regular approximation-relativistic density functional theory at BP86/QZ4P//BP86/TZ2P. ACA values were computed for the methyl, ethyl, i-propyl and t-butyl cations and compared with the corresponding proton affinities (PA). One purpose of this work is to provide an intrinsically consistent set of values of the 298 K ACA of all anionic (XH (n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (group 18) along the periods 1-6. Another purpose is to determine and rationalize the trend in affinity for a cation as the latter varies from proton to t-butyl cation. This undertaking is supported by quantitative bond energy decomposition analyses. Correlations are established between PA and ACA values. PMID:20882538

  17. Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

    PubMed Central

    Veldhuis, Gertjan; Broos, Jaap; Poolman, Bert; Scheek, Ruud M.

    2005-01-01

    Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with KD-values in the nano- and micromolar range, respectively. However, reported KD-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in KD-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeIImtl. Furthermore, the affinity of EnzymeIImtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeIImtl in the same range as mannitol. PMID:15879478

  18. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  19. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  20. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  1. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  2. Chasing polys: Interdisciplinary affinity and its connection to physics identity

    NASA Astrophysics Data System (ADS)

    Scott, Tyler D.

    This research is based on two motivations that merge by means of the frameworks of interdisciplinary affinity and physics identity. First, a goal of education is to develop interdisciplinary abilities in students' thinking and work. But an often ignored factor is students interests and beliefs about being interdisciplinary. Thus, this work develops and uses a framework called interdisciplinary affinity. It encompasses students interests in making connections across disciplines and their beliefs about their abilities to make those connections. The second motivation of this research is to better understand how to engage more students with physics. Physics identity describes how a student sees themselves in relation to physics. By understanding how physics identity is developed, researchers and educators can identify factors that increase interest and engagement in physics classrooms. Therefore, physics identity was used in conjunction with interdisciplinary affinity. Using a mixed methods approach, this research used quantitative data to identify the relationships interdisciplinary affinity has with physics identity and the physics classroom. These connections were explored in more detail using a case study of three students in a high school physics class. Results showed significant and positive relationships between interdisciplinary affinity and physics identity, including the individual interest and recognition components of identity. It also identified characteristics of physics classrooms that had a significant, positive relationship with interdisciplinary affinity. The qualitative case study highlighted the importance of student interest to the relationship between interdisciplinary affinity and physics identity. It also identified interest and mastery orientation as key to understanding the link between interdisciplinary affinity and the physics classroom. These results are a positive sign that by understanding interdisciplinary affinity and physics identity

  3. Enzyme-gold affinity labelling of cellulose.

    PubMed

    Berg, R H; Erdos, G W; Gritzali, M; Brown, R D

    1988-04-01

    The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Optimal affine-invariant matching: performance characterization

    NASA Astrophysics Data System (ADS)

    Costa, Mauro S.; Haralick, Robert M.; Shapiro, Linda G.

    1992-04-01

    The geometric hashing scheme proposed by Lamdan and Wolfson can be very efficient in a model-based matching system, not only in terms of the computational complexity involved, but also in terms of the simplicity of the method. In a recent paper, we discussed errors that can occur with this method due to quantization, stability, symmetry, and noise problems. These errors make the original geometric hashing technique unsuitable for use on the factory floor. Beginning with an explicit noise model, which the original Lamdan and Wolfson technique lacks, we derived an optimal approach that overcomes these problems. We showed that the results obtained with the new algorithm are clearly better than the results from the original method. This paper addresses the performance characterization of the geometric hashing technique, more specifically the affine-invariant point matching, applied to the problem of recognizing and determining the pose of sheet metal parts. The experiments indicate that with a model having 10 to 14 points, with 2 points of the model undetected and 10 extraneous points detected, and with the model points perturbed by Gaussian noise of standard deviation 3 (0.58 of range), the average amount of computation required to obtain an answer is equivalent to trying 11 of the possible three-point bases. The misdetection rate, measured by the percentage of correct bases matches that fail to verify, is 0.9. The percentage of incorrect bases that successfully produced a match that did verify (false alarm rate) is 13. And, finally, 2 of the experiments failed to find a correct match and verify it. Results for experiments with real images are also presented.

  5. Selectively Promiscuous Opioid Ligands: Discovery of High Affinity/Low Efficacy Opioid Ligands with Substantial Nociceptin Opioid Peptide Receptor Affinity

    PubMed Central

    2015-01-01

    Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for μ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo. PMID:24761755

  6. Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

    PubMed

    Benkoussa, M; Nominé, B; Mouchon, A; Lefebvre, B; Bernardon, J M; Formstecher, P; Lefebvre, P

    1997-01-01

    The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

  7. Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

    PubMed

    Zhao, Wei-Wei; Liu, Fu-Feng; Shi, Qing-Hong; Sun, Yan

    2014-09-12

    In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. PMID:25064536

  8. How Structure Defines Affinity in Protein-Protein Interactions

    PubMed Central

    Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

    2014-01-01

    Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

  9. Analysis of biomolecular interactions using affinity microcolumns: a review.

    PubMed

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L; White, Christopher J; Carter, NaTasha; Hage, David S

    2014-10-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  10. Value Added?

    ERIC Educational Resources Information Center

    UCLA IDEA, 2012

    2012-01-01

    Value added measures (VAM) uses changes in student test scores to determine how much "value" an individual teacher has "added" to student growth during the school year. Some policymakers, school districts, and educational advocates have applauded VAM as a straightforward measure of teacher effectiveness: the better a teacher, the better students…

  11. Affine differential geometry analysis of human arm movements.

    PubMed

    Flash, Tamar; Handzel, Amir A

    2007-06-01

    Humans interact with their environment through sensory information and motor actions. These interactions may be understood via the underlying geometry of both perception and action. While the motor space is typically considered by default to be Euclidean, persistent behavioral observations point to a different underlying geometric structure. These observed regularities include the "two-thirds power law", which connects path curvature with velocity, and "local isochrony", which prescribes the relation between movement time and its extent. Starting with these empirical observations, we have developed a mathematical framework based on differential geometry, Lie group theory and Cartan's moving frame method for the analysis of human hand trajectories. We also use this method to identify possible motion primitives, i.e., elementary building blocks from which more complicated movements are constructed. We show that a natural geometric description of continuous repetitive hand trajectories is not Euclidean but equi-affine. Specifically, equi-affine velocity is piecewise constant along movement segments, and movement execution time for a given segment is proportional to its equi-affine arc-length. Using this mathematical framework, we then analyze experimentally recorded drawing movements. To examine movement segmentation and classification, the two fundamental equi-affine differential invariants-equi-affine arc-length and curvature are calculated for the recorded movements. We also discuss the possible role of conic sections, i.e., curves with constant equi-affine curvature, as motor primitives and focus in more detail on parabolas, the equi-affine geodesics. Finally, we explore possible schemes for the internal neural coding of motor commands by showing that the equi-affine framework is compatible with the common model of population coding of the hand velocity vector when combined with a simple assumption on its dynamics. We then discuss several alternative explanations

  12. Diacylglycerol kinase η1 is a high affinity isozyme for diacylglycerol.

    PubMed

    Komenoi, Suguru; Takemura, Fumika; Sakai, Hiromichi; Sakane, Fumio

    2015-05-01

    Diacylglycerol kinase (DGK) η plays important roles in various patho-physiological events such as oncogenesis. In this study, we performed an enzymological characterization of DGKη splice variant 1 (DGKη1). The Km value for diacylglycerol was 0.14 mol%. Intriguingly, the Km value of DGKη1 for diacylglycerol was at least 9-fold lower than those of other DGK isozymes including DGKα, indicating that DGKη1 is a high affinity isozyme for diacylglycerol. Therefore, DGKη1 is a unique DGK isozyme, which may function at particular membrane sites where only low concentrations of diacylglycerol are supplied.

  13. Affinity- and topology-dependent bound on current fluctuations

    NASA Astrophysics Data System (ADS)

    Pietzonka, Patrick; Barato, Andre C.; Seifert, Udo

    2016-08-01

    We provide a proof of a recently conjectured universal bound on current fluctuations in Markovian processes. This bound establishes a link between the fluctuations of an individual observable current, the cycle affinities driving the system into a non-equilibrium steady state, and the topology of the network. The proof is based on a decomposition of the network into independent cycles with both positive affinity and positive stationary cycle current. This formalism allows for a refinement of the bound for systems in equilibrium or with locally vanishing affinities.

  14. Affinity+: Semi-Structured Brainstorming on Large Displays

    SciTech Connect

    Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.; LaMothe, Ryan R.; Endert, Alexander

    2013-04-27

    Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

  15. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  16. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  17. ANALYSIS OF DRUG INTERACTIONS WITH HIGH DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Sike; Sobansky, Matthew R.; Hage, David S.

    2009-01-01

    Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography. This approach was evaluated by using it to examine the binding of high density lipoprotein (HDL) to the drugs propranolol or verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPLC columns with reproducible binding to propranolol over four to five days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interactions were occurring between R/S-propranolol and HDL at 37°C: saturable binding with an association equilibrium constant (Ka) of 1.1–1.9 × 105 M−1, and non-saturable binding with an overall affinity constant (n Ka) of 3.7–4.1 × 104 M−1. Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a Ka of 6.0 × 104 M−1 at 37°C for the saturable sites and a n Ka value for the non-saturable sites of 2.5 × 104 M−1. These measured affinities gave good agreement with solution-phase values. The results indicated HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body. PMID:19833090

  18. Theoretical and Experimental Determination of the Proton Affinity of (CF3CH2)2O

    NASA Technical Reports Server (NTRS)

    Zehe, Michael J.; Ball, David W.

    1998-01-01

    We report the experimental determination of the proton affinity of the molecule (CF3CH2)2O using chemical ionization mass spectrometry, and we compare it to the theoretical value obtained for protonation at the oxygen atom using the calculational methodology (MP2/6-31G**//MP2/3-21G). The proton affinity for this molecule as measured by bracketing experiments was between 724 kJ/mole and 741 kJ/mole. Ab initio (MP2/6-31G**//MP2/3-21G) calculations yield a value of about 729 kJ/mole, in agreement with the chemical ionization experiments. The results of these and related calculations suggest that the (MP2/6-31G**//MP2/3-21G) methodology is acceptable for estimating the proton affinities of partially-and fully-fluorinated methyl and ethyl ethers. We submit that any conclusions about the chemistry of fluoroether polymer lubricants based on their basicity can also be predicted reliably with such calculations.

  19. Pre-Yield Non-Affine Fluctuations and A Hidden Critical Point in Strained Crystals

    PubMed Central

    Das, Tamoghna; Ganguly, Saswati; Sengupta, Surajit; Rao, Madan

    2015-01-01

    A crystalline solid exhibits thermally induced localised non-affine droplets in the absence of external stress. Here we show that upon an imposed shear, the size of these droplets grow until they percolate at a critical strain, well below the value at which the solid begins to yield. This critical point does not manifest in most thermodynamic or mechanical properties, but is hidden and reveals itself in the onset of inhomogeneities in elastic moduli, marked changes in the appearance and local properties of non-affine droplets and a sudden enhancement in defect pair concentration. Slow relaxation of stress and an-elasticity appear as observable dynamical consequences of this hidden criticality. Our results may be directly verified in colloidal crystals with video microscopy techniques but are expected to have more general validity. PMID:26039380

  20. Electron affinities of d1 transition metal chloride clusters and onset of super halogen behavior

    NASA Astrophysics Data System (ADS)

    Behera, Swayamprabha; Joseph, Jorly; Jena, Purusottam

    2011-03-01

    Geometry, electronic structure, and electron affinity of d1 transition metal chloride clusters (MCl n , M = Sc,Y, La; n = 1--5) have been calculated using density functional theory. Chlorine atoms are chemically bound in all cases except for MCl 5 . The electron affinities of MCl n (n = 1--3) are small and increase only marginally as a function of n until the valence of the metal atom is consumed. Beyond this, they rise sharply and reach a value of 5.96, 6.03 and 5.90 eV for ScCl 4 , YCl 4 and LaCl 4 , respectively and remain high for n = 5. MCl n , (n = 4,5) clusters, therefore, behave as superhalogens. Results are compared with available experimental data

  1. Electron affinities of d1 transition metal chloride clusters and onset of super halogen behavior

    NASA Astrophysics Data System (ADS)

    Joseph, Jorly; Behera, Swayamprabha; Jena, Purusottam

    2010-09-01

    Geometry, electronic structure, and electron affinity of d1 transition metal chloride clusters (MCl n, M = Sc, Y, La; n = 1-5) have been calculated using density functional theory. Chlorine atoms are chemically bound in all cases except for MCl 5. The electron affinities of MCl n ( n = 1-3) are small and increase only marginally as a function of n until the valence of the metal atom is consumed. Beyond this, they rise sharply and reach a value of 5.96, 6.03 and 5.90 eV for ScCl 4, YCl 4 and LaCl 4, respectively and remain high for n = 5. MCl n, ( n = 4,5) clusters, therefore, behave as superhalogens. Results are compared with available experimental data.

  2. Self-Affinity, Self-Similarity and Disturbance of Soil Seed Banks by Tillage.

    PubMed

    Dias, Luís S

    2013-07-05

    Soil seed banks were sampled in undisturbed soil and after soil had been disturbed by tillage (tine, harrow or plough). Seeds were sorted by size and shape, and counted. Size-number distributions were fitted by power law equations that allowed the identification of self-similarity and self-affinity. Self-affinity and thus non-random size-number distribution prevailed in undisturbed soil. Self-similarity and thus randomness of size-number distribution prevailed after tillage regardless of the intensity of disturbance imposed by cultivation. The values of fractal dimensions before and after tillage were low, suggesting that short-term, short-range factors govern size-number distribution of soil seed banks.

  3. Superconformal Chern-Simons partition functions of affine D-type quiver from Fermi gas

    NASA Astrophysics Data System (ADS)

    Moriyama, Sanefumi; Nosaka, Tomoki

    2015-09-01

    We consider the partition function of the superconformal Chern-Simons theories with the quiver diagram being the affine D-type Dynkin diagram. Rewriting the partition function into that of a Fermi gas system, we show that the perturbative expansions in 1 /N are summed up to an Airy function, as in the ABJM theory or more generally the theories of the affine A-type quiver. As a corollary, this provides a proof for the previous proposal in the large N limit. For special values of the Chern-Simons levels, we further identify three species of the membrane instantons and also conjecture an exact expression of the overall constant, which corresponds to the constant map in the topological string theory. [Figure not available: see fulltext.

  4. Method for resurrecting negative electron affinity photocathodes after exposure to an oxidizing gas

    DOEpatents

    Mulhollan, Gregory A; Bierman, John C

    2012-10-30

    A method by which negative electron affinity photocathodes (201), single crystal, amorphous, or otherwise ordered, can be made to recover their quantum yield following exposure to an oxidizing gas has been discovered. Conventional recovery methods employ the use of cesium as a positive acting agent (104). In the improved recovery method, an electron beam (205), sufficiently energetic to generate a secondary electron cloud (207), is applied to the photocathode in need of recovery. The energetic beam, through the high secondary electron yield of the negative electron affinity surface (203), creates sufficient numbers of low energy electrons which act on the reduced-yield surface so as to negate the effects of absorbed oxidizing atoms thereby recovering the quantum yield to a pre-decay value.

  5. Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration affinity membrane.

    PubMed

    Konovalova, Viktoriia; Guzikevich, Kateryna; Burban, Anatoliy; Kujawski, Wojciech; Jarzynka, Karolina; Kujawa, Joanna

    2016-11-01

    In order to prepare ultrafiltration membranes possessing biocatalytic properties, α-amylase has been immobilized on cellulose membranes. Enzyme immobilization was based on a covalent bonding between chitosan and a surface of cellulose membrane, followed by an attachment of Cibacron Blue F3G-A dye as affinity ligand. Various factors affecting the immobilization process, such as enzyme concentration, pH of modifying solution, zeta-potential of membrane surface, and stability of immobilized enzyme were studied. The applicability of immobilized α-amylase has been investigated in ultrafiltration processes. The immobilization of α-amylase on membrane surface allows to increase the value of mass transfer coefficient and to decrease the concentration polarization effect during ultrafiltration of starch solutions. The enzyme layer on the membrane surface prevents a rapid increase of starch concentration due to the amylase hydrolysis of starch in the boundary layer. The presented affinity immobilization technique allows also for the regeneration of membranes from inactivated enzyme.

  6. KW-3902, a selective high affinity antagonist for adenosine A1 receptors.

    PubMed Central

    Nonaka, H.; Ichimura, M.; Takeda, M.; Kanda, T.; Shimada, J.; Suzuki, F.; Kase, H.

    1996-01-01

    1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively. PMID:8732272

  7. Community Development, Transitional Value, and Institutional Affinity: Outdoor Orientation Program Impacts

    ERIC Educational Resources Information Center

    Howard, Ryan A.; O'Connell, Timothy S.; Lathrop, Anna H.

    2016-01-01

    This article examines the impact of an outdoor orientation program (OOP) on a cohort of first-year university students who participated in a canoe trip facilitated by peer leaders. The curriculum included training for outdoor skills and transitional guidance to university life (i.e., strategies for time management, critical thinking, becoming…

  8. Dense Stereo Matching Method Based on Local Affine Model.

    PubMed

    Li, Jie; Shi, Wenxuan; Deng, Dexiang; Jia, Wenyan; Sun, Mingui

    2013-07-01

    A new method for constructing an accurate disparity space image and performing an efficient cost aggregation in stereo matching based on local affine model is proposed in this paper. The key algorithm includes a new self-adapting dissimilarity measurement used for calculating the matching cost and a local affine model used in cost aggregation stage. Different from the traditional region-based methods, which try to change the matching window size or to calculate an adaptive weight to do the aggregation, the proposed method focuses on obtaining the efficient and accurate local affine model to aggregate the cost volume while preserving the disparity discontinuity. Moreover, the local affine model can be extended to the color space. Experimental results demonstrate that the proposed method is able to provide subpixel precision disparity maps compared with some state-of-the-art stereo matching methods. PMID:24163727

  9. On the thermodynamic basis of the affinity decay rate

    NASA Astrophysics Data System (ADS)

    Garcia-Colín, L. S.; Piña, E.; de la Selva, S. M. T.

    1990-03-01

    In the past five years exhaustive studies in chemical reactions have lead to an empirical equation describing how isothermal-isometric homogeneous reactions evolve towards equilibrium independently of their particular mechanism or rate law. Such an equation expresses the time rate of change of the chemical affinity as a linear function of the inverse of time. In this paper we show that by invoking the local equilibrium hypothesis one may provide, a time evolution equation for the chemical affinity that is uniquely given by the solution of the particular rate law of the reaction considered. Consequently such an equation is not of the same functional form for all reactions. On the other hand, integration of Dalton's law under specific initial conditions, together with the local equilibrium assumption and the ideality requirement for the reacting species, exhibits a unique inverse time decay for the chemical affinity. This explains the good fitting of the inverse in time dependence of the chemical affinity with experimental data.

  10. A thermodynamic approach to the affinity optimization of drug candidates.

    PubMed

    Freire, Ernesto

    2009-11-01

    High throughput screening and other techniques commonly used to identify lead candidates for drug development usually yield compounds with binding affinities to their intended targets in the mid-micromolar range. The affinity of these molecules needs to be improved by several orders of magnitude before they become viable drug candidates. Traditionally, this task has been accomplished by establishing structure activity relationships to guide chemical modifications and improve the binding affinity of the compounds. As the binding affinity is a function of two quantities, the binding enthalpy and the binding entropy, it is evident that a more efficient optimization would be accomplished if both quantities were considered and improved simultaneously. Here, an optimization algorithm based upon enthalpic and entropic information generated by Isothermal Titration Calorimetry is presented.

  11. Frontal affinity chromatography (FAC): theory and basic aspects.

    PubMed

    Kasai, Ken-ichi

    2014-01-01

    Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory. PMID:25117240

  12. Bidirectional Elastic Image Registration Using B-Spline Affine Transformation

    PubMed Central

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C.; Wang, Chen; Kaminski, Naftali; Pu, Jiantao

    2014-01-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-Spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bi-directional instead of the traditional unidirectional objective / cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy. PMID:24530210

  13. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  14. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

    PubMed

    Cozzi, Nicholas V; Daley, Paul F

    2016-02-01

    correlations among physicochemical properties of the congeneric 5-substituted-DALT compounds. The descriptors included electronic (σp), hydrophobic (π), and steric (CMR) parameters. The binding affinity at 5-HT1A, 5-HT1D, 5-HT7, and κ opioid receptors was positively correlated with the steric volume parameter CMR. At α2A, α2B, and α2C receptors, and at the histamine H1 receptor, binding affinity was correlated with the Hammett substituent parameter σp; higher affinity was associated with larger σp values. At the σ2 receptor, higher affinity was correlated with increasing π. These correlations should aid in the development of more potent and selective drugs within this family of compounds.

  15. Hydride affinities of cumulated, isolated, and conjugated dienes in acetonitrile.

    PubMed

    Zhu, Xiao-Qing; Liang, Hao; Zhu, Yan; Cheng, Jin-Pei

    2008-11-01

    The hydride affinities (defined as the enthalpy changes in this work) of 15 polarized dienes [five phenyl sulfone substituted allenes (1a), the corresponding five isolated dienes (1b), and the corresponding five conjugated dienes (1c)] in acetonitrile solution were determined by titration calorimetry for the first time. The results display that the hydride affinity scales of the 15 dienes in acetonitrile range from -71.6 to -73.9 kcal/mol for 1a, from -46.2 to -49.7 kcal/mol for 1b, and from -45.0 to -46.5 kcal/mol for 1c, which indicates that the hydride-obtaining abilities of the cumulated dienes (1a) are not only much larger than those of the corresponding conjugated dienes (1c) but also much larger than those of the corresponding isolated dienes (1b). The hydrogen affinities of the 15 dienes as well as the hydrogen affinities and the proton affinities of the radical anions of the dienes (1(-*)) in acetonitrile were also evaluated by using relative thermodynamic cycles according to Hess's law. The results show that (i) the hydrogen affinities of the neutral dienes 1 cover a range from -44.5 to -45.6 kcal/mol for 1a, from -20.4 to -21.4 kcal/mol for 1b, and from -17.3 to -18.5 kcal/mol for 1c; (ii) the hydrogen affinities of the radical anions of the dienes (1(-*)) in acetonitrile cover a range from -40.6 to -47.2 kcal/mol for 1a(-*), from -21.6 to -29.6 kcal/mol for 1b(-*), and from -10.0 to -15.4 kcal/mol for 1c(-*); (iii) the proton affinities of the 15 1a(-*) in acetonitrile cover a range from -97.0 to -100.6 kcal/mol for 1a(-*), from -77.8 to -83.4 kcal/mol for 1b(-*), and from -66.2 to -68.9 kcal/mol for 1c(-*). The main reasons for the great difference between the cumulated dienes and the corresponding isolated and conjugated dienes in the hydride affinity, hydrogen affinity, and proton affinity have been examined. It is evident that these experimental results should be quite valuable to facilitate the elucidation of the origins of the especially high

  16. Flexible Linker Modulates Glycosaminoglycan Affinity of Decorin Binding Protein A.

    PubMed

    Morgan, Ashli; Sepuru, Krishna Mohan; Feng, Wei; Rajarathnam, Krishna; Wang, Xu

    2015-08-18

    Decorin binding protein A (DBPA) is a glycosaminoglycan (GAG)-binding adhesin found on the surface of the bacterium Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. DBPA facilitates bacterial adherence to extracellular matrices of human tissues and is crucial during the early stage of the infection process. Interestingly, DBPA from different strains (B31, N40, and PBr) show significant differences in GAG affinities, but the structural basis for the differences is not clear. In this study, we show that GAG affinity of N40 DBPA is modulated in part by flexible segments that control access to the GAG binding site, such that shortening of the linker leads to higher GAG affinity when analyzed using ELISA, gel mobility shift assay, solution NMR, and isothermal titration calorimetry. Our observation that GAG affinity differences among different B. burgdorferi strains can be attributed to a flexible linker domain regulating access to the GAG-binding domain is novel. It also provides a rare example of how neutral amino acids and dynamic segments in GAG binding proteins can have a large influence on GAG affinity and provides insights into why the number of basic amino acids in the GAG-binding site may not be the only factor determining GAG affinity of proteins. PMID:26223367

  17. The Prostaglandin Transporter: Eicosanoid Reuptake, Control of Signaling, and Development of High-Affinity Inhibitors as Drug Candidates

    PubMed Central

    Schuster, Victor L.; Chi, Yuling; Lu, Run

    2015-01-01

    We discovered the prostaglandin transporter (PGT) and cloned the human cDNA and gene. PGT transports extracellular prostaglandins (PGs) into the cytoplasm for enzymatic inactivation. PGT knockout mice have elevated prostaglandin E2 (PGE2) and neonatal patent ductus arteriosus, which reflects PGT's control over PGE2 signaling at EP1/EP4 cell-surface receptors. Interestingly, rescued PGT knockout pups have a nearly normal phenotype, as do human PGT nulls. Given the benign phenotype of PGT genetic nulls, and because PGs are useful medicines, we have approached PGT as a drug target. Triazine library screening yielded a lead compound of inhibitory constant 50% (IC50) = 3.7 μM, which we developed into a better inhibitor of IC50 378 nM. Further structural improvements have yielded 26 rationally designed derivatives with IC50 < 100 nM. The therapeutic approach of increasing endogenous PGs by inhibiting PGT offers promise in diseases such as pulmonary hypertension and obesity. PMID:26330684

  18. Identification and affinity of very high affinity binding sites for the phenylalkylamine series of Ca/sup +/ channel blockers in the Drosophila nervous system

    SciTech Connect

    Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M.; Berge, J.B.; Lazdunski, M.

    1987-10-06

    The interaction of putative Ca/sup 2 +/ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-(/sup 3/H)D888 and (+/-)-(/sup 3/H)verapamil. These ligands recognize a single class of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a K/sub d/ value as exceptional low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca/sup 2 +/ channel blockers as well as bepridil inhibited (-)-(/sup 3/H)D888 binding in a competitive way with K/sub d/ values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor was used in photoaffinity experiments. A protein of M/sub r/ 135,000 +/- 5000 was specifically labeled after ultraviolet irradiation.

  19. CHARACTERIZATION OF THE BINDING OF SULFONYLUREA DRUGS TO HSA BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions. PMID:20435530

  20. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics.

    PubMed

    Aweda, Tolulope A; Meares, Claude F

    2012-02-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.

  1. Quantitative structure-activity relationships for polychlorinated hydroxybiphenyl estrogen receptor binding affinity: An assessment of conformer flexibility

    SciTech Connect

    Bradbury, S.P.; Ankley, G.T.; Mekenyan, O.G.

    1996-11-01

    A diverse group of xenobiotics has a high binding affinity to the estrogen receptor (ER), suggesting that it can accommodate large variability in ligand structure. Relationships between xenobiotic surface, binding affinity, and estrogenic response have been suggested to be dependent on the conformational structures of the ligands. To explore the influence of conformational flexibility on ER binding affinity, a quantitative structure-activity relationship (QSAR) study was undertaken with estradiol, diethylstilbestrol, and a set of polychlorinated hydroxybiphenyls (PCHBs) of environmental concern. Although the low-energy minima of the PCHB congeners suggested that interconversions among conformers were likely, the electronic parameters associated with the conformer geometries for a specific PCHB congener could vary significantly. The results of the QSAR analysis suggested that among the PCHBs studied, the most polarizable conformers (lower absolute volume polarizability values) were most closely associated with ER binding affinity. Across the set of polarizable conformers, which did not include the low-energy gas-phase conformers, the electron donating properties of the hydroxy moiety and the aromatic component of the estradiol A ring analogue in the PCHBs were found to be correlated with higher ER binding affinity.

  2. Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

    SciTech Connect

    Jagadeesh, G.; Deth, R.C.

    1987-11-01

    Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of (/sup 3/H)prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate (Gpp(NH)p). A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. (/sup 3/H)Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with (/sup 3/H)prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.

  3. Temporal variation in phenetic affinity of early Upper Egyptian male cranial series.

    PubMed

    Keita, S O Y; Boyce, A J

    2008-04-01

    We carried out an exploratory historical biology study using temporally distinguished groups of predynastic-Early Dynastic male crania from the region of Upper Egypt. The objectives were, first, to determine the overall pattern of phenetic affinity between temporally sequential series and in relation to the earliest series and, second, to explore the possible meanings of the pattern of relationship to sociohistorical change. The cranial series were designated early predynastic, late predynastic, terminal predynastic, and Dynasty I. Craniometric phenetic affinity was ascertained using Mahalanobis distances; a 5% level of probability was chosen for significance. The distance matrix values were ordered into hierarchies of dissimilarity from each series (distance hierarchies) and tabulated for time-successive groups, including the temporally earliest series (i.e., serialized by time). The principal observations were as follows. The overall pattern was not one in which the values between all series were statistically insignificant; nor was it one of consistent sequential increase of biological distance from the earliest series. There was a notable and statistically significant distance between the early and late predynastic groups, with the late and terminal predynastic groups mutually having the lowest and statistically insignificant distances with each other. The value between the terminal predynastic and Dynasty I series was generally larger than the values between other groups and was statistically significant. The overall pattern is possibly consistent with archeological interpretations that postulate increasing intraregional interactions during the late and terminal predynastic periods and the rise of an Egyptian state that eventually included northern Egypt. PMID:18720900

  4. Analysis of Aged Human Serum Albumin Affinity for Doxazosin.

    PubMed

    Chudzik, Mariola; Równicka-Zubik, Joanna; Pożycka, Jadwiga; Pawelczak, Bartosz; Sulkowska, Anna

    2016-01-01

    Structural changes of human serum albumin (HSA) caused by old age and coexisting diseases result in differences in the binding of doxazosin (DOX). DOX is a postsynaptic α1- adrenoreceptor antagonist used for treatment of hypertension and benign prostatic hyperplasia. In elderly people suffering from various renal or hepatic diseases the significant portion of N-form of human serum albumin (normal) is converted to A-form (aged). The differences in binding of doxazosin to N- and Aform of albumin are an important factor, which may determines therapeutic dosage and toxicity of the test drug. To indicate these differences, the technique of fluorescence spectroscopy was used. The association constant (Ka) obtained from fluorescence quenching demonstrated that doxazosin has higher affinity for AHSA than for HSA. In order to describe the cooperativity in binding process, the values of the Hill's coefficient has been analysed. For DOX-HSA system (λex 295 nm) Hill's coefficient is close to 1 and it indicates that there is a single class of binding sites. For DOX-HSA (λex 275 nm) and DOX-AHSA (λex 275 nm and λex 295 nm) systems we observed positive cooperativity (nH>1). A greater red shift of fluorescence emission maximum of AHSA than HSA in the presence of DOX was observed. This suggests that the binding of DOX to AHSA was accompanied by a stronger increase in polarity around the fluorophores in comparison to HSA. The binding interaction between DOX and HSA has been also studied by molecular docking simulation.

  5. Value Added

    ERIC Educational Resources Information Center

    Wilson, M. Roy

    2015-01-01

    With more than a thousand honors programs or colleges in the United States and that number growing every year, defining the value of honors is a significant undertaking. Honors seems to have become an obligatory upgrade that no college or university president can afford to be without, but there is more than institutional trending to be considered,…

  6. Value Added

    ERIC Educational Resources Information Center

    Welch, Matt

    2004-01-01

    This article profiles retiring values teacher Gene Doxey and describes his foundational contributions to the students of California's Ramona Unified School District. Every one of the Ramona Unified School District's 7,200 students is eventually funneled through Doxey's Contemporary Issues class, a required rite of passage between elementary school…

  7. Estimation of enthalpy data for reactions involving gas phase ions utilizing lattice potential energies: fluoride ion affinities (FIA) and pF- values of mSbF5(l) and mSbF5(g) (m = 1, 2, 3), AsF5(g), AsF5.SO2(c). Standard enthalpies of formation: Delta(f)H degrees (SbmF5m+1)(-),g) (m = 1, 2, 3), Delta(f)H degrees (AsF6(-),g), and Delta(f)H degrees (NF4+,g).

    PubMed

    Jenkins, H Donald Brooke; Roobottom, H K; Passmore, Jack

    2003-05-01

    Fluoride ion affinity (FIA) values (and the associated pF(-) values) are difficult to establish experimentally for pentafluorides of arsenic and antimony. Our approach, utilizing estimated lattice potential energies, provides a further opportunity to establish this data for liquid (and gaseous) SbF(5) and gaseous AsF(5) which compliments values obtained using ab initio routes for monomeric gas phase molecules and adds to results based on rigorous methods. A strategy is developed whereby construction of (multiple) Born-Fajans-Haber cycles centered around the (target) FIA reaction of interest yield a plethora of estimates for the enthalpy change of interest. This general approach is illustrated here by specific estimation of some experimentally based FIA values of SbF(5) and AsF(5). FIA values/kJ mol(-1) and pF- values estimated in this paper are FIA(SbF(5),l) approximately equal to -475 (+/-63), pF-(SbF(5),l) = 11.4 (+/-1.5); FIA(SbF(5),g) approximately equal to -506 (+/-63), pF-(SbF(5),g) = 12.4 (+/-1.5); FIA(2SbF(5),l) approximately equal to -609 (+/-63), pF- (2SbF(5),l) = 14.6 (+/-1.5); FIA (2SbF(5),g) approximately equal to -671 (+/-63), pF- (2SbF(5),g) = 16.0 (+/-1.5); FIA (3SbF(5),l) approximately -635 (+/-39), pF(-) (3SbF(5),l) = 15.2 (+/-0.9); FIA(3SbF(5),g) approximately -728 (+/-39), pF(-) (3SbF(5),g) = 17.4 (+/-0.9); FIA(AsF(5),g) approximately equal to -421 (+/-22), pF(-) (AsF(5),g) = 10.1 (+/- 0.5); and FIA (AsF(5).SO(2),s) approximately equal to -390 (+/-22), pF(-) (AsF(5).SO(2),s) = 9.3 (+/-0.5). Related standard enthalpies of formation (in kJ mol(-1)) are also assigned: Delta(f)H degrees (SbF(6)(-),g) approximately equal to -2075 (+/-52); Delta(f)H degrees (Sb(2)F(11)(-),g) approximately equal to -3520 (+/-63); Delta(f)H degrees (Sb(3)F(16)(-),g) approximately equal to -4874 (+/-39); Delta(f)H degrees (NF(4)(+),g) approximately equal to 903 (+/-32); Delta(f)H degrees (AsF(6)(-),g) approximately equal to -1907 (+/-22).

  8. Binding of ionic species: a general approach to measuring binding constants and assessing affinities.

    PubMed

    Roelens, Stefano; Vacca, Alberto; Venturi, Chiara

    2009-03-01

    Bound together: The association of receptors with ionic species cannot be assimilated to the binding of neutral guests. When dealing with salts, both ion pairing and binding to the free and the ion-paired ionic guest determine the actual association pattern (see figure). The general issue of measuring association constants and assessing affinities for ions is addressed and validated in two cases of anion binding.A general approach to the largely underestimated issue of measuring binding constants and assessing affinities in the binding of ionic species is described. The approach is based on a rigorous, nongraphical determination of binding constants in multiequilibrium systems by nonlinear regression of chemical shift data from NMR titrations and on the use of the BC(50) descriptor for assessing affinities and ranking the binding ability of receptors on a common scale. The approach has been validated with two tripodal anion-binding receptors, namely, a ureidic (1) and a pyrrolic (2) receptor, binding to tetramethylammonium chloride in CDCl(3)/CD(3)CN (80:20). A set of five and six formation constants could be measured for 1 and 2, respectively, including, in addition to the ion pair, complexes of the free and the ion-paired anion. The BC(50) values calculated from the measured constants allowed a quantitative assessment of each receptor's binding affinity towards the chloride anion, the pyrrolic receptor showing a 15-fold larger affinity over the ureidic receptor, a figure that quantifies the improvement obtained by replacing the amido-pyrrolic for ureidic binding groups on the tripodal scaffold of the receptor. The results have shown that, in contrast to common practice, neither of the two systems could be appropriately described by a 1:1 association with the anion only, but required the ion-pairing and ion-pair binding equilibria to be taken into account because these contribute substantially to the complexation process. The BC(50) descriptor has also been shown

  9. Erythrocyte 2,3-DPG, ATP and oxygen affinity in hemodialysis patients.

    PubMed

    Ninness, J R; Kimber, R W; McDonald, J W

    1974-10-01

    Patients on a chronic hemodialysis regimen were studied with respect to their erythrocyte adaptation to anemia. Erythrocyte 2,3-diphosphoglycerate (DPG) concentration was suboptimal compared with that of anemic patients who were not uremic. In uremic patients erythrocyte 2,3-DPG correlated poorly with hemoglobin level but more strongly with plasma pH. Differences between observed levels of erythrocyte 2,3-DPG and the values predicted using data from other anemic patients also correlated with pH. Gradual correction of plasma pH with oral sodium bicarbonate resulted in a substantial increase in erythrocyte 2,3-DPG and a decrease in oxygen affinity. Therefore, maintenance of normal pH in uremic subjects may improve tissue oxygenation. On the other hand, the rapid correction of acidosis during dialysis resulted in increased oxygen affinity. This response was due to the direct effect of pH on oxygen affinity in the absence of a significant change in erythrocyte 2,3-DPG or adenosine triphosphate (ATP) during hemodialysis. Erythrocyte ATP but not 2,3-DPG correlated with serum inorganic phosphate in uremic subjects. A 21% reduction of serum phosphate produced by ingestion of aluminum hydroxide gel had no significant effect on these variables.

  10. An SF1 affinity model to identify branch point sequences in human introns

    PubMed Central

    Pastuszak, Alexander W.; Joachimiak, Marcin P.; Blanchette, Marco; Rio, Donald C.; Brenner, Steven E.; Frankel, Alan D.

    2011-01-01

    Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117 499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3′-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing. PMID:21071404

  11. Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains.

    PubMed

    Vidal, M; Liu, W-Q; Lenoir, C; Salzmann, J; Gresh, N; Garbay, C

    2004-06-15

    This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.

  12. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  13. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  14. Comparative investigation of surface transfer doping of hydrogen terminated diamond by high electron affinity insulators

    NASA Astrophysics Data System (ADS)

    Verona, C.; Ciccognani, W.; Colangeli, S.; Limiti, E.; Marinelli, Marco; Verona-Rinati, G.

    2016-07-01

    We report on a comparative study of transfer doping of hydrogenated single crystal diamond surface by insulators featured by high electron affinity, such as Nb2O5, WO3, V2O5, and MoO3. The low electron affinity Al2O3 was also investigated for comparison. Hole transport properties were evaluated in the passivated hydrogenated diamond films by Hall effect measurements, and were compared to un-passivated diamond films (air-induced doping). A drastic improvement was observed in passivated samples in terms of conductivity, stability with time, and resistance to high temperatures. The efficiency of the investigated insulators, as electron accepting materials in hydrogenated diamond surface, is consistent with their electronic structure. These surface acceptor materials generate a higher hole sheet concentration, up to 6.5 × 1013 cm-2, and a lower sheet resistance, down to 2.6 kΩ/sq, in comparison to the atmosphere-induced values of about 1 × 1013 cm-2 and 10 kΩ/sq, respectively. On the other hand, hole mobilities were reduced by using high electron affinity insulator dopants. Hole mobility as a function of hole concentration in a hydrogenated diamond layer was also investigated, showing a well-defined monotonically decreasing trend.

  15. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  16. Ethylene binding site affinity in ripening apples

    SciTech Connect

    Blankenship, S.M. . Dept. of Horticultural Science); Sisler, E.C. )

    1993-09-01

    Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

  17. [Affinity of the elements in group VI of the periodic table to tumors and organs].

    PubMed

    Ando, A; Hisada, K; Ando, I

    1976-10-01

    In order to investigate the tumor affinity radioisotopes, chromium (51Cr), molybdenum (99Mo), tungsten (181W), selenium (75Se) and tellurium (127mTe)--the elements of group VI in the periodic table--were examined, using the rats which were subcutaneously transplanted with Yoshida sarcoma. Seven preprarations, sodium chromate (Na251CrO4), chromium chloride (51CrCl3), normal ammonium molybdate ((NH4)299MoO7), sodium tungstate (Na2181WO4), sodium selenate (Na275SeO4), sodium selenite (Na275SeO3) and tellurous acid (H2127mTeO3) were injected intravenously to each group of tumor bearing rats. These rats were sacrificed at various periods after injection of each preparation: 3 hours, 24 hours and 48 hours in all preparations. The radioactivities of the tumor, blood, muscle, liver, kidney and spleen were measured by a well-type scintillation counter, and retention values (in every tissue including the tumor) were calculated in percent of administered dose per g-tissue weight. All of seven preparations did not have any affinity for malignant tumor. Na251CrO4 and H2127mTeO3 had some affinity for the kidneys, and Na275SeO3 had some affinity for the liver. Na2181WO4 and (NH4)299MoO4 disappeared very rapidly from the blood and soft tissue, and about seventy-five percent of radioactivity was excreted in urine within first 3 hours.

  18. Proton affinity changes driving unidirectional proton transport in the bacteriorhodopsin photocycle.

    PubMed

    Onufriev, Alexey; Smondyrev, Alexander; Bashford, Donald

    2003-10-01

    Bacteriorhodopsin is the smallest autonomous light-driven proton pump. Proposals as to how it achieves the directionality of its trans-membrane proton transport fall into two categories: accessibility-switch models in which proton transfer pathways in different parts of the molecule are opened and closed during the photocycle, and affinity-switch models, which focus on changes in proton affinity of groups along the transport chain during the photocycle. Using newly available structural data, and adapting current methods of protein protonation-state prediction to the non-equilibrium case, we have calculated the relative free energies of protonation microstates of groups on the transport chain during key conformational states of the photocycle. Proton flow is modeled using accessibility limitations that do not change during the photocycle. The results show that changes in affinity (microstate energy) calculable from the structural models are sufficient to drive unidirectional proton transport without invoking an accessibility switch. Modeling studies for the N state relative to late M suggest that small structural re-arrangements in the cytoplasmic side may be enough to produce the crucial affinity change of Asp96 during N that allows it to participate in the reprotonation of the Schiff base from the cytoplasmic side. Methodologically, the work represents a conceptual advance compared to the usual calculations of pK(a) using macroscopic electrostatic models. We operate with collective states of protonation involving all key groups, rather than the individual-group pK(a) values traditionally used. When combined with state-to-state transition rules based on accessibility considerations, a model for non-equilibrium proton flow is obtained. Such methods should also be applicable to other active proton-transport systems. PMID:14499620

  19. Gradient-based habitat affinities predict species vulnerability to drought.

    PubMed

    Debinski, Diane M; Caruthers, Jennet C; Cook, Dianne; Crowley, Jason; Wickham, Hadley

    2013-05-01

    Ecological fingerprints of climate change are becoming increasingly evident at broad geographical scales as measured by species range shifts and changes in phenology. However, finer-scale species-level responses to environmental fluctuations may also provide an important bellwether of impending future community responses. Here we examined changes in abundance of butterfly species along a hydrological gradient of six montane meadow habitat types in response to drought. Our data collection began prior to the drought, and we were able to track changes for 11 years, of which eight were considered mild to extreme drought conditions. We separated the species into those that had an affinity for hydric vs. xeric habitats. We suspected that drought would favor species with xeric habitat affinities, but that there could be variations in species-level responses along the hydrological gradient. We also suspected that mesic meadows would be most sensitive to drought conditions. Temporal trajectories were modeled for both species groups (hydric vs. xeric affinity) and individual species. Abundances of species with affinity for xeric habitats increased in virtually all meadow types. Conversely, abundances of species with affinity for hydric habitats decreased, particularly in mesic and xeric meadows. Mesic meadows showed the most striking temporal abundance trajectory: Increasing abundances of species with xeric habitat affinity were offset by decreasing or stable abundances of species with hydric habitat affinity. The one counterintuitive finding was that, in some hydric meadows, species with affinity for hydric habitats increased. In these cases, we suspect that decreasing moisture conditions in hydric meadows actually increased habitat suitability because sites near the limit of moisture extremes for some species became more acceptable. Thus, species responses were relatively predictable based upon habitat affinity and habitat location along the hydrological gradient, and

  20. Natural mutations change the affinity of μ-theraphotoxin-Hhn2a to voltage-gated sodium channels.

    PubMed

    Zhang, Fan; Liu, Yu; Zhang, Changxin; Li, Jing; Yang, Zuqin; Gong, Xue; Gan, Yunxiang; Chen, Ping; Liu, Zhonghua; Liang, Songping

    2015-01-01

    μ-Theraphotoxin-Hhn2a (HNTX-III) isolated from the venom of the spider Ornithoctonus hainana is a selective antagonist of neuronal tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channels (VGSCs). Intriguingly, previous transcriptomic study revealed HNTX-III family consists of more than 15 precursors, in which the 20(th) and 24(th) residues of the mature sequences are variable. Try20 and Ser24 of HNTX-III are mutated to His20 and Asn24 of other members, respectively. In addition, the alkaline residue His26 of the potent VGSC inhibitor HNTX-III is substituted by acidic residue Asp of the weak VGSC inhibitor HNTX-I. Therefore, four mutants of HNTX-III, HNTX-III-Y20H, -S24N, -H26D and -Y20H/24N, were synthesized to examine the effects of these natural mutations on the inhibitory activity of HNTX-III. They were subjected to an electrophysiological screening on five VGSC subtypes (Nav1.3-1.5, Nav1.7 and Nav1.8) expressed on HEK293 cells by whole-cell patch clamp. Like HNTX-III, all mutants only displayed inhibitory activity on Nav1.3 and Nav1.7 among the five subtypes, but the inhibitory potency was much lower than that of HNTX-III. Regarding Nav1.7, the IC50 values of HNTX-III-Y20H, -S24N, -H26D and -Y20H/S24N were increased by approximately 62-, 8.4-, 49- and 19.5-folds compared with that of HNTX-III, respectively. Similar data were obtained for Nav1.3. Our results provide new insights into the activity-related residues of HNTX-III at genic level. Furthermore, the reduced potency of the four mutants probably reflects natural selection might favor and reserve the most potent bioactivity of HNTX-III which is one of the most abundant fractions of the venom. PMID:25447770

  1. Melanin affinity and its possible role in neurodegeneration.

    PubMed

    Karlsson, Oskar; Lindquist, Nils Gunnar

    2013-12-01

    Certain drugs with melanin affinity are known to have caused pigmentary lesions in the eye and skin. This was the basis for the hypothesis that compounds with melanin affinity may cause damage also in other melanin-bearing tissues such as the substantia nigra. The heterogeneity of compounds that binds to melanin is large. Toxins, drugs, and several other compounds have melanin affinity. Compounds showing the highest affinity are mainly organic amines and metal ions. The binding of toxicants to melanin probably protects the cells initially. However, the binding is normally, slowly reversible and melanin may accumulate the toxicant and gradually release it into the cytosol. Several studies indicate that neuromelanin may play a significant role both in the initiation and in the progression of neurodegeneration. MPTP/MPP(+) that has been causally linked with Parkinsonism has high affinity for neuromelanin, and the induced dopaminergic denervation correlates with the neuromelanin content in the cells. This shows that the toxicological implications of the accumulation of toxicants in pigmented neurons and its possible role in neurodegeneration should not be neglected. Extracellular neuromelanin has been reported to activate dendritic cells and microglia. An initial neuronal damage induced by a neurotoxicant that leaks neuromelanin from the cells may therefore lead to a vicious cycle of neuroinflammation and further neurodegeneration. Although there are many clues to the particular vulnerability of dopaminergic neurons of substantia nigra in Parkinson's disease, the critical factors are not known. Further studies to determine the importance of neuromelanin in neurodegeneration and Parkinson's disease are warranted.

  2. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%. PMID:26856529

  3. Improving antibody binding affinity and specificity for therapeutic development.

    PubMed

    Bostrom, Jenny; Lee, Chingwei V; Haber, Lauric; Fuh, Germaine

    2009-01-01

    Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

  4. Crystal Structure of A Plant Dual-Affinity Nitrate Transporter

    PubMed Central

    Sun, Ji; Bankston, John R.; Payandeh, Jian; Hinds, Thomas R.; Zagotta, William N.; Zheng, Ning

    2014-01-01

    Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter, which can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr101. Yet how this posttranslational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 indeed dimerizes in the cell membrane and the phosphomimetic mutation of Thr101 converts the protein into a monophasic high affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes. PMID:24572362

  5. Fluorogen-Activating-Proteins as Universal Affinity Biosensors for Immunodetection

    PubMed Central

    Gallo, Eugenio; Vasilev, Kalin V.; Jarvik, Jonathan

    2014-01-01

    Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins –Protein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. PMID:24122476

  6. Affinity Purification of Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Kadonaga, James T.; Tjian, Robert

    1986-08-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.

  7. Learning a peptide-protein binding affinity predictor with kernel ridge regression

    PubMed Central

    2013-01-01

    Background The cellular function of a vast majority of proteins is performed through physical interactions with other biomolecules, which, most of the time, are other proteins. Peptides represent templates of choice for mimicking a secondary structure in order to modulate protein-protein interaction. They are thus an interesting class of therapeutics since they also display strong activity, high selectivity, low toxicity and few drug-drug interactions. Furthermore, predicting peptides that would bind to a specific MHC alleles would be of tremendous benefit to improve vaccine based therapy and possibly generate antibodies with greater affinity. Modern computational methods have the potential to accelerate and lower the cost of drug and vaccine discovery by selecting potential compounds for testing in silico prior to biological validation. Results We propose a specialized string kernel for small bio-molecules, peptides and pseudo-sequences of binding interfaces. The kernel incorporates physico-chemical properties of amino acids and elegantly generalizes eight kernels, comprised of the Oligo, the Weighted Degree, the Blended Spectrum, and the Radial Basis Function. We provide a low complexity dynamic programming algorithm for the exact computation of the kernel and a linear time algorithm for it’s approximation. Combined with kernel ridge regression and SupCK, a novel binding pocket kernel, the proposed kernel yields biologically relevant and good prediction accuracy on the PepX database. For the first time, a machine learning predictor is capable of predicting the binding affinity of any peptide to any protein with reasonable accuracy. The method was also applied to both single-target and pan-specific Major Histocompatibility Complex class II benchmark datasets and three Quantitative Structure Affinity Model benchmark datasets. Conclusion On all benchmarks, our method significantly (p-value ≤ 0.057) outperforms the current state-of-the-art methods at predicting

  8. AB-Bind: Antibody binding mutational database for computational affinity predictions.

    PubMed

    Sirin, Sarah; Apgar, James R; Bennett, Eric M; Keating, Amy E

    2016-02-01

    Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high-affinity and high-specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody-Bind (AB-Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB-Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16-0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < -1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction. PMID:26473627

  9. Afi-Chip: An Equipment-Free, Low-Cost, and Universal Binding Ligand Affinity Evaluation Platform.

    PubMed

    Song, Yanling; Shi, Yuanzhi; Li, Xingrui; Ma, Yanli; Gao, Mingxuan; Liu, Dan; Mao, Yu; Zhu, Zhi; Lin, Hui; Yang, Chaoyong

    2016-08-16

    Binding affinity characterization is of great importance for aptamer screening because the dissociation constant (Kd) value is a key parameter for evaluating molecular interaction. However, conventional methods often require sophisticated equipment and time-consuming processing. Here, we present a portable device, Afi-Chip, as an equipment-free, rapid, low-cost, and universal platform for evaluation of the aptamer affinity. The Afi-Chip displays a distance readout based on the reaction of an enzyme catalyzing the decomposition of H2O2 for gas generation to push the movement of ink bar. Taking advantage of translating the recognition signal to distance signal and realizing the regents mixing and quantitative readout on the chip, we successfully monitored the aptamer evolution process and characterized binding affinity of aptamers against multiple types of targets, including small molecule glucose, cancer biomarker protein EpCAM, and tumor cell SW620. We also applied the Afi-Chip for rapid characterization of the affinity between anti-HCG and HCG to demonstrate the generality for the molecular interaction study. All of the Kd values obtained are comparable to those reported in the literature or obtained by sophisticated instruments such as a flow cytometer. The Afi-Chip offers a new approach for equipment-free investigation of molecular interactions, such as aptamer identification, ligand selection monitoring, and drug screening. PMID:27454185

  10. Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

    PubMed

    Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

    2016-03-14

    The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds.

  11. Afi-Chip: An Equipment-Free, Low-Cost, and Universal Binding Ligand Affinity Evaluation Platform.

    PubMed

    Song, Yanling; Shi, Yuanzhi; Li, Xingrui; Ma, Yanli; Gao, Mingxuan; Liu, Dan; Mao, Yu; Zhu, Zhi; Lin, Hui; Yang, Chaoyong

    2016-08-16

    Binding affinity characterization is of great importance for aptamer screening because the dissociation constant (Kd) value is a key parameter for evaluating molecular interaction. However, conventional methods often require sophisticated equipment and time-consuming processing. Here, we present a portable device, Afi-Chip, as an equipment-free, rapid, low-cost, and universal platform for evaluation of the aptamer affinity. The Afi-Chip displays a distance readout based on the reaction of an enzyme catalyzing the decomposition of H2O2 for gas generation to push the movement of ink bar. Taking advantage of translating the recognition signal to distance signal and realizing the regents mixing and quantitative readout on the chip, we successfully monitored the aptamer evolution process and characterized binding affinity of aptamers against multiple types of targets, including small molecule glucose, cancer biomarker protein EpCAM, and tumor cell SW620. We also applied the Afi-Chip for rapid characterization of the affinity between anti-HCG and HCG to demonstrate the generality for the molecular interaction study. All of the Kd values obtained are comparable to those reported in the literature or obtained by sophisticated instruments such as a flow cytometer. The Afi-Chip offers a new approach for equipment-free investigation of molecular interactions, such as aptamer identification, ligand selection monitoring, and drug screening.

  12. Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

    PubMed

    Chen, Bin; Li, Rong; Li, Shiyu; Chen, Xiaoli; Yang, Kaidi; Chen, Guoliang; Ma, Xiaoxun

    2016-02-01

    The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors. PMID:26632098

  13. Photodetachment of electrons from phosphide ion - The electron affinity of PH2.

    NASA Technical Reports Server (NTRS)

    Smyth, K. C.; Brauman, J. I.

    1972-01-01

    Measurement of the relative cross section for photodetachment of electrons from PH2(-) in the wavelength region 725 to 1020 nm (1.71 to 1.22 eV). An ion cyclotron resonance spectrometer was used to generate, trap, and detect the negative ions, and two light sources were employed to study photodetachment: a 1000-W xenon arc lamp with a grating monochromator and a continuously tunable laser. A single sharp threshold in the cross-section curve was observed, and a detailed analysis yielded an electron affinity value of 1.25 plus or minus 0.03 eV.

  14. Specific capture of uranyl protein targets by metal affinity chromatography.

    PubMed

    Basset, Christian; Dedieu, Alain; Guérin, Philippe; Quéméneur, Eric; Meyer, Daniel; Vidaud, Claude

    2008-03-28

    To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis. PMID:18308325

  15. High affinity ligands from in vitro selection: Complex targets

    PubMed Central

    Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

    1998-01-01

    Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

  16. Enhancing Community Detection By Affinity-based Edge Weighting Scheme

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    Community detection refers to an important graph analytics problem of finding a set of densely-connected subgraphs in a graph and has gained a great deal of interest recently. The performance of current community detection algorithms is limited by an inherent constraint of unweighted graphs that offer very little information on their internal community structures. In this paper, we propose a new scheme to address this issue that weights the edges in a given graph based on recently proposed vertex affinity. The vertex affinity quantifies the proximity between two vertices in terms of their clustering strength, and therefore, it is ideal for graph analytics applications such as community detection. We also demonstrate that the affinity-based edge weighting scheme can improve the performance of community detection algorithms significantly.

  17. Recent advances in affinity capillary electrophoresis for binding studies.

    PubMed

    Albishri, Hassan M; El Deeb, Sami; AlGarabli, Noura; AlAstal, Raghda; Alhazmi, Hassan A; Nachbar, Markus; El-Hady, Deia Abd; Wätzig, Hermann

    2014-01-01

    The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry. PMID:25534793

  18. On the electron affinity of the oxygen atom

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Langhoff, S. R.; Partridge, H.; Taylor, P. R.

    1986-01-01

    The electron affinity of oxygen is computed to be 1.287 eV, at the full CI level using a 6s5p3d 2f Slater-type orbital basis and correlating only the 2p electrons. The best CASSCF-MRCI result including only 2p correlation is 1.263 eV. However, inclusion of 2s intrashell and 2s2p intershell coorelation increases the computed EA to 1.290 eV at the CASSCF-MRCI level. At the full CI basis set limit, the 2s contribution to the electron affinity is estimated to be as large as 0.1 eV. The higher excitation contribution to the electron affinity is found to increase substantially with basis set completeness, especially when the 2s electrons are correlated. Relativistic effects are shown to make a small (less than 0.01 eV) change in the EA.

  19. On the electron affinity of the oxygen atom

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Langhoff, S. R.; Partridge, H.; Taylor, P. R.

    1986-01-01

    The electron affinity (EA) of oxygen is computed to be 1.287 eV, using 2p electron full configuration-interaction (CI) wave functions expanded in a 6s5p3d2f Slater-type orbital basis. The best complete active space self-consistent field - multireference CI (CASSCF-MRCI) result including only 2p correlation is 1.263 eV. However, inclusion of 2s intrashell and 2s2p intershell correlation increases the computed EA to 1.290 at the CASSCF-MRCI level. At the full CI basis set limit, the 2s contribution to the electron affinity is estimated to be as large as 0.1 eV. This study clearly establishes the synergistic effect between the higher excitations and basis set completeness on the electron affinity when the 2s electrons are correlated.

  20. Latest technologies for the enhancement of antibody affinity.

    PubMed

    Wark, Kim L; Hudson, Peter J

    2006-08-01

    High affinity antibodies are crucial both for the discovery and validation of biomarkers for human health and disease and as clinical diagnostic and therapeutic reagents. This review describes some of the latest technologies for the design, mutation and selection of high affinity antibodies that provide a paradigm for molecular evolution of a far wider range of proteins including enzymes. Strategies include both in vivo and in vitro methods and embrace the latest concepts for antibody display and selection. Specifically, affinity enhancement can be tailored to the target-binding surface, typically the complementary determining region (CDR) loops in antibodies, whereas enhanced stability, expression or catalytic properties can be affected by selected changes to the core protein scaffold. Together, these technologies provide a rapid and powerful strategy to drive the next generation of protein-based reagents for numerous clinical, environmental and agribusiness applications.

  1. Oxygen affinity of blood in altitude Sherpas.

    PubMed

    Samaja, M; Veicsteinas, A; Cerretelli, P

    1979-08-01

    Oxygen equilibrium curves on blood within 6 h from sampling have been estimated from polarographic measurements of oxyhemoglobin concentration, in 13 male 14- to 50-yr old Sherpas residing at 3,850 m above sea level (Kumjung, Nepal). In samples with red blood cell counts = 4.7 +/- 0.8 (SD) x 10(6)/mm3, total hemoglobin concentration [Hb] = 17.0 +/- 1.9 g/dl, and hematocrit = 53.3 +/- 5.0, the mean oxygen half-saturation of hemoglobin (P50) (pH = 7.4 and PCO2 = 40 Torr) was 27.3 +/- 1.8 Torr. The P50 of altitude Sherpas was not significantly different from that of acclimatized lowlanders (28.2 +/- 1.3; n = 7), sea-level Caucasian residents (26.5 +/- 1.0; n = 17), and Sherpas at sea level (27.1; n = 3). The 2,3-diphosphoglyceric acid-to-hemoglobin concentration ratio ([2,3-DPG]/[Hb]) in altitude Sherpas was 1.22 +/- 0.03, the same as that of acclimatized Caucasians (1.22 +/- 0.10). The Bohr effect measured for the blood of one altitude Sherpas by the ratio deltalog P50/deltapH was -0.32 and -0.45 at PCO2 levels of 40 and 20 Torr, respectively. These values are not significantly different from those found in Caucasians at sea level where deltalog P50/deltalpH was -0.35 and -0.42, respectively. It is concluded that the P50 in native highlanders is not significantly different from that observed in sea-level dwellers. [2,3-DPG]/[Hb] at altitude, both in natives and in newcomers, is 20% higher than in sea-level residents.

  2. Affine generalization of the Komar complex of general relativity

    NASA Astrophysics Data System (ADS)

    Mielke, Eckehard W.

    2001-02-01

    On the basis of the ``on shell'' Noether identities of the metric-affine gauge approach of gravity, an affine superpotential is derived which comprises the energy- and angular-momentum content of exact solutions. In the special case of general relativity (GR) or its teleparallel equivalent, the Komar or Freud complex, respectively, are recovered. Applying this to the spontaneously broken anti-de Sitter gauge model of McDowell and Mansouri with an induced Euler term automatically yields the correct mass and spin of the Kerr-AdS solution of GR with a (induced) cosmological constant without the factor two discrepancy of the Komar formula.

  3. Considering affinity: an ethereal conversation (part two of three).

    PubMed

    Winsor, Mary P

    2015-06-01

    In 1840 Hugh Strickland published a diagram showing the relationships of genera of birds in the kingfisher family. Three years later he applied this mapping idea to genera of birds of prey and songbirds, creating a large wall chart that he displayed to colleagues but never published. Both of his diagrams featured a scale of degrees of affinity. The meaning of taxonomic affinity was something Darwin thought about deeply. Details in the chart undermine Strickland's claim that his method was purely inductive. PMID:25547607

  4. A quantum affine algebra for the deformed Hubbard chain

    NASA Astrophysics Data System (ADS)

    Beisert, Niklas; Galleas, Wellington; Matsumoto, Takuya

    2012-09-01

    The integrable structure of the one-dimensional Hubbard model is based on Shastry's R-matrix and the Yangian of a centrally extended \\mathfrak {sl}(2|2) superalgebra. Alcaraz and Bariev have shown that the model admits an integrable deformation whose R-matrix has recently been found. This R-matrix is of trigonometric type and here we derive its underlying exceptional quantum affine algebra. We also show how the algebra reduces to the above-mentioned Yangian and to the conventional quantum affine \\mathfrak {sl}(2|2) algebra in two special limits.

  5. The metrizability problem for Lorentz-invariant affine connections

    NASA Astrophysics Data System (ADS)

    Urban, Zbyněk; Volná, Jana

    2016-07-01

    The invariant metrizability problem for affine connections on a manifold, formulated by Tanaka and Krupka for connected Lie groups actions, is considered in the particular cases of Lorentz and Poincaré (inhomogeneous Lorentz) groups. Conditions under which an affine connection on the open submanifold ℝ × (ℝ3\\{(0, 0, 0)}) of the Euclidean space ℝ4 coincides with the Levi-Civita connection of some SO(3, 1), respectively (ℝ4 × sSO(3, 1))-invariant metric field are studied. We give complete description of metrizable Lorentz-invariant connections. Explicit solutions (metric fields) of the invariant metrizability equations are found and their properties are discussed.

  6. Dynamic output feedback H ∞ control for affine fuzzy systems

    NASA Astrophysics Data System (ADS)

    Wang, Huimin; Yang, Guang-Hong

    2013-06-01

    This article investigates the problem of designing H ∞ dynamic output feedback controllers for nonlinear systems, which are described by affine fuzzy models. The system outputs have been chosen as premise variables, which can guarantee that the plant and the controller always switch to the same region. By using a piecewise Lyapunov function and adding slack matrix variables, a piecewise-affine dynamic output feedback controller design method is obtained in the formulation of linear matrix inequalities (LMIs), which can be efficiently solved numerically. In contrast to the existing work, the proposed approach needs less LMI constraints and leads to less conservatism. Finally, numerical examples illustrate the effectiveness of the new result.

  7. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  8. Affinity Adsorbents Based on Carriers Activated by Epoxy-compounds

    NASA Astrophysics Data System (ADS)

    Klyashchitskii, B. A.; Kuznetsov, P. V.

    1984-10-01

    The review is devoted to the synthesis and applications of affinity adsorbents based on carriers activated by epoxy-compounds. The methods for the introduction of epoxy-groups into carriers of different chemical types are discussed and conditions for the immobilisation of three-dimensional spacers and low-molecular-weight and polymeric ligands on carriers containing epoxy-groups are considered. Data are presented on the properties and applications of adsorbents of this type in affinity chromatography. The bibliography includes 144 references.

  9. and as Vertex Operator Extensionsof Dual Affine Algebras

    NASA Astrophysics Data System (ADS)

    Bowcock, P.; Feigin, B. L.; Semikhatov, A. M.; Taormina, A.

    We discover a realisation of the affine Lie superalgebra and of the exceptional affine superalgebra as vertex operator extensions of two algebras with ``dual'' levels (and an auxiliary level-1 algebra). The duality relation between the levels is . We construct the representation of on a sum of tensor products of , , and modules and decompose it into a direct sum over the spectral flow orbit. This decomposition gives rise to character identities, which we also derive. The extension of the construction to is traced to the properties of embeddings into and their relation with the dual pairs. Conversely, we show how the representations are constructed from representations.

  10. Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle

    SciTech Connect

    Madison, J.M.; Jones, C.A.; Tom-Moy, M.; Brown, J.K.

    1987-03-01

    Muscarinic cholinergic receptors have been classified into subtypes based on their high (M-1 subtype) or low (M-2 subtype) affinities for the nonclassic antagonist pirenzepine, and this classification has important experimental and therapeutic implications. Because muscarinic receptors are abundant in the airways where they mediate several different cellular responses, the goal of this study was to characterize the affinities of pirenzepine for the muscarinic receptors in bovine tracheal mucosa and smooth muscle. After isolating membrane particulates from mucosa and smooth muscle, as well as from bovine cerebral cortex (a known source of M-1 receptors), we used /sup 3/H-quinuclidinyl benzilate to label muscarinic receptors in the particulates and performed competition radioligand binding assays in the presence of either atropine or pirenzepine. Receptors from all 3 tissues (mucosa, smooth muscle, and cerebral cortex) were of a relatively uniform affinity for atropine (range of KI values: 0.8 +/- 0.4 X 10(-9) to 2.4 +/- 1.7 X 10(-9) M), as would be predicted for this classic muscarinic antagonist. By contrast, affinities for pirenzepine differed depending on the tissue. In cerebral cortex, the majority of receptors were of high affinity for pirenzepine (KI = 1.8 +/- 1.4 X 10(-8) M). In both mucosa and smooth muscle, receptors were of low affinity for pirenzepine (Kl = 4.8 +/- 0.4 to 6.9 +/- 3.8 X 10(-7) M). We conclude that muscarinic cholinergic receptors in bovine tracheal mucosa and smooth muscle are predominantly of the M-2 subtype.

  11. Valuing Stillbirths.

    PubMed

    Phillips, John; Millum, Joseph

    2015-07-01

    Estimates of the burden of disease assess the mortality and morbidity that affect a population by producing summary measures of health such as quality-adjusted life years (QALYs) and disability-adjusted life years (DALYs). These measures typically do not include stillbirths (fetal deaths occurring during the later stages of pregnancy or during labor) among the negative health outcomes they count. Priority-setting decisions that rely on these measures are therefore likely to place little value on preventing the more than three million stillbirths that occur annually worldwide. In contrast, neonatal deaths, which occur in comparable numbers, have a substantial impact on burden of disease estimates and are commonly seen as a pressing health concern. In this article we argue in favor of incorporating unintended fetal deaths that occur late in pregnancy into estimates of the burden of disease. Our argument is based on the similarity between late-term fetuses and newborn infants and the assumption that protecting newborns is important. We respond to four objections to counting stillbirths: (1) that fetuses are not yet part of the population and so their deaths should not be included in measures of population health; (2) that valuing the prevention of stillbirths will undermine women's reproductive rights; (3) that including stillbirths implies that miscarriages (fetal deaths early in pregnancy) should also be included; and (4) that birth itself is in fact ethically significant. We conclude that our proposal is ethically preferable to current practice and, if adopted, is likely to lead to improved decisions about health spending.

  12. Valuing Stillbirths.

    PubMed

    Phillips, John; Millum, Joseph

    2015-07-01

    Estimates of the burden of disease assess the mortality and morbidity that affect a population by producing summary measures of health such as quality-adjusted life years (QALYs) and disability-adjusted life years (DALYs). These measures typically do not include stillbirths (fetal deaths occurring during the later stages of pregnancy or during labor) among the negative health outcomes they count. Priority-setting decisions that rely on these measures are therefore likely to place little value on preventing the more than three million stillbirths that occur annually worldwide. In contrast, neonatal deaths, which occur in comparable numbers, have a substantial impact on burden of disease estimates and are commonly seen as a pressing health concern. In this article we argue in favor of incorporating unintended fetal deaths that occur late in pregnancy into estimates of the burden of disease. Our argument is based on the similarity between late-term fetuses and newborn infants and the assumption that protecting newborns is important. We respond to four objections to counting stillbirths: (1) that fetuses are not yet part of the population and so their deaths should not be included in measures of population health; (2) that valuing the prevention of stillbirths will undermine women's reproductive rights; (3) that including stillbirths implies that miscarriages (fetal deaths early in pregnancy) should also be included; and (4) that birth itself is in fact ethically significant. We conclude that our proposal is ethically preferable to current practice and, if adopted, is likely to lead to improved decisions about health spending. PMID:25395144

  13. Water properties in fern spores: sorption characteristics relating to water affinity, glassy states, and storage stability.

    PubMed

    Ballesteros, Daniel; Walters, Christina

    2007-01-01

    Ex situ conservation of ferns may be accomplished by maintaining the viability of stored spores for many years. Storage conditions that maximize spore longevity can be inferred from an understanding of the behaviour of water within fern spores. Water sorption properties were measured in spores of five homosporeous species of ferns and compared with properties of pollen, seeds, and fern leaf tissue. Isotherms were constructed at 5, 25, and 45 degrees C and analysed using different physicochemical models in order to quantify chemical affinity and heat (enthalpy) of sorption of water in fern spores. Fern spores hydrate slowly but dry rapidly at ambient relative humidity. Low Brunauer-Emmet-Teller monolayer values, few water-binding sites according to the D'Arcy-Watt model, and limited solute-solvent compatibility according to the Flory-Huggins model suggest that fern spores have low affinity for water. Despite the low water affinity, fern spores demonstrate relatively high values of sorption enthalpy (DeltaH(sorp)). Parameters associated with binding sites and DeltaH(sorp) decrease with increasing temperature, suggesting temperature- and hydration-dependent changes in volume of spore macromolecules. Collectively, these data may relate to the degree to which cellular structures within fern spores are stabilized during drying and cooling. Water sorption properties within fern spores suggest that storage at subfreezing temperatures will give longevities comparable with those achieved with seeds. However, the window of optimum water contents for fern spores is very narrow and much lower than that measured in seeds, making precise manipulation of water content imperative for achieving maximum longevity.

  14. Batroxobin binds fibrin with higher affinity and promotes clot expansion to a greater extent than thrombin.

    PubMed

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2013-06-01

    Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ' variant with an extended γ-chain. Thrombin binds to the γ'-chain and forms a higher affinity interaction with γA/γ'-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 μM) even though it does not interact with the γ'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 μM, the α(17-51) and Bβ(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties. PMID:23612970

  15. High-affinity nasal extraction of vinyl acetate vapor is carboxylesterase dependent.

    PubMed

    Bogdanffy, M S; Manning, L A; Sarangapani, R

    1999-10-01

    Vinyl acetate induces nasal tumors in rats, but not mice. Species differences in airflow patterns, physiology, and biochemistry complicate extrapolation of nasal dosimetry from rats to humans. Physiologically based pharmacokinetic modeling of vinyl acetate dosimetry in rats suggested the presence of a saturable metabolic removal pathway in rat nasal mucus. We explored the possibility that this pathway is either a cytochrome P-450 2E1 (CYP2E1) or high-affinity carboxylesterase. Nasal extraction of vinyl acetate vapor (150 ppm) was measured in the surgically isolated nasal cavity of anesthetized rats. Vinyl acetate (150 ppm) was extracted with 73% efficiency in controls. Pretreatment of rats with the CYP2E1 inhibitor diallyl sulfide (DAS) had no effect on extraction, despite significantly reducing CYP2E1 activity. Pretreatment with bis(p-nitrophenyl) phosphate (BNPP), a carboxylesterase inhibitor, reduced extraction to approximately 41%. Acetaldehyde production was similarly unaffected by DAS but was reduced to 55% of control by BNPP. Rat nasal mucus carboxylesterase activity had a K(m) value (32 microM) similar, within a factor of 2, to the value predicted by the physiologically based model, although V(max) was significantly lower than the model prediction. Histochemical observations support the inference that the high-affinity carboxylesterase is bound to the luminal plasma membrane of nasal tissue and is not readily released by nasal lavage, providing an explanation for the low V(max) of the lavage enzyme. This high-affinity isoenzyme could be important in the removal of odorants from the sensory cell-rich nasal olfactory epithelium.

  16. Modeling the binding affinity of structurally diverse industrial chemicals to carbon using the artificial intelligence approaches.

    PubMed

    Gupta, Shikha; Basant, Nikita; Rai, Premanjali; Singh, Kunwar P

    2015-11-01

    Binding affinity of chemical to carbon is an important characteristic as it finds vast industrial applications. Experimental determination of the adsorption capacity of diverse chemicals onto carbon is both time and resource intensive, and development of computational approaches has widely been advocated. In this study, artificial intelligence (AI)-based ten different qualitative and quantitative structure-property relationship (QSPR) models (MLPN, RBFN, PNN/GRNN, CCN, SVM, GEP, GMDH, SDT, DTF, DTB) were established for the prediction of the adsorption capacity of structurally diverse chemicals to activated carbon following the OECD guidelines. Structural diversity of the chemicals and nonlinear dependence in the data were evaluated using the Tanimoto similarity index and Brock-Dechert-Scheinkman statistics. The generalization and prediction abilities of the constructed models were established through rigorous internal and external validation procedures performed employing a wide series of statistical checks. In complete dataset, the qualitative models rendered classification accuracies between 97.04 and 99.93%, while the quantitative models yielded correlation (R(2)) values of 0.877-0.977 between the measured and the predicted endpoint values. The quantitative prediction accuracies for the higher molecular weight (MW) compounds (class 4) were relatively better than those for the low MW compounds. Both in the qualitative and quantitative models, the Polarizability was the most influential descriptor. Structural alerts responsible for the extreme adsorption behavior of the compounds were identified. Higher number of carbon and presence of higher halogens in a molecule rendered higher binding affinity. Proposed QSPR models performed well and outperformed the previous reports. A relatively better performance of the ensemble learning models (DTF, DTB) may be attributed to the strengths of the bagging and boosting algorithms which enhance the predictive accuracies. The

  17. The role of the 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs. I: Antagonist correlation analysis.

    PubMed

    Fiorella, D; Rabin, R A; Winter, J C

    1995-10-01

    Investigations conducted over the past 3 decades have demonstrated that serotonergic receptors, specifically the 5-HT2A and 5-HT2C subtypes, play an important role in the behavioral effects of hallucinogenic compounds. The present study was designed to determine the respective significance of these two receptors in the stimulus effects of LSD and (-)DOM in the rat. Specifically, the interactions of a series of serotonergic antagonists (risperidone, pirenpirone, metergoline, ketanserin, loxapine, LY53857, pizotyline, spiperone, cyprohepatadine, mesulergine, promethazine, and thioridazine) with the LSD stimulus and the (-)DOM stimulus in LSD-trained subjects was defined. From these data, IC50 values were determined for the inhibition of the LSD-appropriate responding elicited by either 0.1 mg/kg LSD (15-min pretreatment time) or 0.4 mg/kg (-)DOM (75-min pretreatment). In addition, the affinities of these antagonists for 5-HT2A and 5-HT2C receptors were determined in radioligand competition studies, 5-HT2A affinity correlated significantly with IC50 values for the blockade of the LSD (r = +0.75, P < 0.05) and (-)DOM (r = +0.95, P < 0.001) stimuli in the LSD trained subjects. 5-HT2C affinity did not correlate significantly with either series of IC50 values. These data indicate that (1) the stimulus effects of LSD, and (2) the substitution of (-)DOM for the LSD stimulus are mediated by agonist activity at 5-HT2A receptors.

  18. Surface plasmon resonance reveals a different pattern of proinsulin autoantibodies concentration and affinity in diabetic patients.

    PubMed

    Trabucchi, Aldana; Guerra, Luciano L; Faccinetti, Natalia I; Iacono, Rubén F; Poskus, Edgardo; Valdez, Silvina N

    2012-01-01

    Type 1 diabetes mellitus (DM) is characterized by autoimmune aggression against pancreatic beta cells resulting in absolute deficiency of insulin secretion. The first detectable sign of emerging autoimmunity during the preclinical asymptomatic period is the appearance of diabetes-related autoantibodies. In children at risk for type 1 DM, high-affinity Insulin autoantibodies reactive to proinsulin, are associated with diabetes risk. Autoantibodies are usually measured by radioligand binding assay (RBA) that provides quasi-quantitative values reflecting potency (product between concentration and affinity) of specific autoantibodies. Aiming to improve the characterization of the specific humoral immune response, we selected surface plasmon resonance (SPR) as an alternative method to measure proinsulin autoantibodies (PAA). This novel technology has allowed real time detection of antibodies interaction and kinetic analysis. Herein, we have employed SPR to characterize the PAA present in sera from 28 childhood-onset (mean age 8.31±4.20) and 23 adult-onset diabetic patients (≥65 years old, BMI<30) in terms of concentration and affinity. When evaluating comparatively samples from both groups, childhood-onset diabetic patients presented lower PAA concentrations and higher affinities (median 67.12×10(-9) M and 3.50×10(7) M(-1), respectively) than the adults (median 167.4×10(-9) M and 0.84×10(7) M(-1), respectively). These results are consistent with those from the reference method RBA (Standard Deviation score median 9.49 for childhood-onset group and 5.04 for adult-onset group) where the binding can be directly related to the intrinsic affinity of the antibody, suggesting that there is a different etiopathogenic pathway between both types of clinical presentation of the disease. This technology has shown to be a useful tool for the characterization of PAAs parameters as an alternative to radioimmunoassay, with high versatility and reproducibility associated to low

  19. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  20. "The Hunger Games": Literature, Literacy, and Online Affinity Spaces

    ERIC Educational Resources Information Center

    Curwood, Jen Scott

    2013-01-01

    This article examines adolescent literacy practices related to "The Hunger Games," a young adult novel and the first of a trilogy. By focusing on the interaction of social identities, discourses, and media paratexts within an online affinity space, this ethnographic study offers insight into how young adults engage with contemporary…

  1. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago.

  2. Background correction using dinucleotide affinities improves the performance of GCRMA

    PubMed Central

    Gharaibeh, Raad Z; Fodor, Anthony A; Gibas, Cynthia J

    2008-01-01

    Background High-density short oligonucleotide microarrays are a primary research tool for assessing global gene expression. Background noise on microarrays comprises a significant portion of the measured raw data, which can have serious implications for the interpretation of the generated data if not estimated correctly. Results We introduce an approach to calculate probe affinity based on sequence composition, incorporating nearest-neighbor (NN) information. Our model uses position-specific dinucleotide information, instead of the original single nucleotide approach, and adds up to 10% to the total variance explained (R2) when compared to the previously published model. We demonstrate that correcting for background noise using this approach enhances the performance of the GCRMA preprocessing algorithm when applied to control datasets, especially for detecting low intensity targets. Conclusion Modifying the previously published position-dependent affinity model to incorporate dinucleotide information significantly improves the performance of the model. The dinucleotide affinity model enhances the detection of differentially expressed genes when implemented as a background correction procedure in GeneChip preprocessing algorithms. This is conceptually consistent with physical models of binding affinity, which depend on the nearest-neighbor stacking interactions in addition to base-pairing. PMID:18947404

  3. Affinity through Mathematical Activity: Cultivating Democratic Learning Communities

    ERIC Educational Resources Information Center

    Sengupta-Irving, Tesha

    2014-01-01

    In this article, the author demonstrates how a broader view of what shapes affinity is ideologically and practically linked to creating democratic learning communities. Specifically, the author explores how a teacher employed complex instruction (an equity pedagogy) with her ethnically and racially diverse students in the "lowest track"…

  4. Student Engagement and Neoliberalism: Mapping an Elective Affinity

    ERIC Educational Resources Information Center

    Zepke, Nick

    2015-01-01

    The purpose of this article is to argue that student engagement, an important area for research about learning and teaching in formal higher education, has an elective affinity with neoliberalism, a hegemonic ideology in many countries of the developed world. The paper first surveys an extensive research literature examining student engagement and…

  5. Affinities and beyond! Developing Ways of Seeing in Online Spaces

    ERIC Educational Resources Information Center

    Davies, Julia

    2006-01-01

    This article presents an insider view of an online community of adults involved in sharing digital photography through a host website, Flickr. It describes how reciprocal teaching and learning partnerships in a dynamic multimodal environment are achieved through the creation of a "Third Space" or "Affinity Space", where "Funds of Knowledge" are…

  6. Peptides@mica: from affinity to adhesion mechanism.

    PubMed

    Gladytz, A; John, T; Gladytz, T; Hassert, R; Pagel, M; Risselada, H J; Naumov, S; Beck-Sickinger, A G; Abel, B

    2016-09-14

    Investigating the adsorption of peptides on inorganic surfaces, on the molecular level, is fundamental for medicinal and analytical applications. Peptides can be potent as linkers between surfaces and living cells in biochips or in implantation medicine. Here, we studied the adsorption process of the positively charged pentapeptide RTHRK, a recently identified binding sequence for surface oxidized silicon, and novel analogues thereof to negatively charged mica surfaces. Homogeneous formation of monolayers in the nano- and low micromolar peptide concentration range was observed. We propose an alternative and efficient method to both quantify binding affinity and follow adhesion behavior. This method makes use of the thermodynamic relationship between surface coverage, measured by atomic force microscopy (AFM), and the concomitant free energy of adhesion. A knowledge-based fit to the autocorrelation of the AFM images was used to correct for a biased surface coverage introduced by the finite lateral resolution of the AFM. Binding affinities and mechanisms were further explored by large scale molecular dynamics (MD) simulations. The combination of well validated MD simulations with topological data from AFM revealed a better understanding of peptide adsorption processes on the atomistic scale. We demonstrate that binding affinity is strongly determined by a peptide's ability to form salt bridges and hydrogen bonds with the surface lattice. Consequently, differences in hydrogen bond formation lead to substantial differences in binding affinity despite conservation of the peptide's overall charge. Further, MD simulations give access to relative changes in binding energy of peptide variations in comparison to a lead compound. PMID:27491508

  7. Development of gadolinium based nanoparticles having an affinity towards melanin

    NASA Astrophysics Data System (ADS)

    Morlieras, Jessica; Chezal, Jean-Michel; Miot-Noirault, Elisabeth; Roux, Amandine; Heinrich-Balard, Laurence; Cohen, Richard; Tarrit, Sébastien; Truillet, Charles; Mignot, Anna; Hachani, Roxanne; Kryza, David; Antoine, Rodolphe; Dugourd, Philippe; Perriat, Pascal; Janier, Marc; Sancey, Lucie; Lux, François; Tillement, Olivier

    2013-01-01

    Small Rigid Platforms (SRPs) are sub-5 nanometre gadolinium based nanoparticles that have been developed for multimodal imaging and theranostic applications. They are composed of a polysiloxane network surrounded by gadolinium chelates. A covalent coupling with quinoxaline derivatives has been performed. Such derivatives have proven their affinity for melanin frequently expressed in primary melanoma cases. Three different quinoxaline derivatives have been synthesised and coupled to the nanoparticles. The affinity of the grafted nanoparticles for melanin has then been shown in vitro by surface plasmon resonance on a homemade melanin grafted gold chip.Small Rigid Platforms (SRPs) are sub-5 nanometre gadolinium based nanoparticles that have been developed for multimodal imaging and theranostic applications. They are composed of a polysiloxane network surrounded by gadolinium chelates. A covalent coupling with quinoxaline derivatives has been performed. Such derivatives have proven their affinity for melanin frequently expressed in primary melanoma cases. Three different quinoxaline derivatives have been synthesised and coupled to the nanoparticles. The affinity of the grafted nanoparticles for melanin has then been shown in vitro by surface plasmon resonance on a homemade melanin grafted gold chip. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33457g

  8. Smooth surfaces from bilinear patches: Discrete affine minimal surfaces.

    PubMed

    Käferböck, Florian; Pottmann, Helmut

    2013-06-01

    Motivated by applications in freeform architecture, we study surfaces which are composed of smoothly joined bilinear patches. These surfaces turn out to be discrete versions of negatively curved affine minimal surfaces and share many properties with their classical smooth counterparts. We present computational design approaches and study special cases which should be interesting for the architectural application.

  9. Properties of an affine transport equation and its holonomy

    NASA Astrophysics Data System (ADS)

    Vines, Justin; Nichols, David A.

    2016-10-01

    An affine transport equation was used recently to study properties of angular momentum and gravitational-wave memory effects in general relativity. In this paper, we investigate local properties of this transport equation in greater detail. Associated with this transport equation is a map between the tangent spaces at two points on a curve. This map consists of a homogeneous (linear) part given by the parallel transport map along the curve plus an inhomogeneous part, which is related to the development of a curve in a manifold into an affine tangent space. For closed curves, the affine transport equation defines a "generalized holonomy" that takes the form of an affine map on the tangent space. We explore the local properties of this generalized holonomy by using covariant bitensor methods to compute the generalized holonomy around geodesic polygon loops. We focus on triangles and "parallelogramoids" with sides formed from geodesic segments. For small loops, we recover the well-known result for the leading-order linear holonomy (˜ Riemann × area), and we derive the leading-order inhomogeneous part of the generalized holonomy (˜ Riemann × area^{3/2}). Our bitensor methods let us naturally compute higher-order corrections to these leading results. These corrections reveal the form of the finite-size effects that enter into the holonomy for larger loops; they could also provide quantitative errors on the leading-order results for finite loops.

  10. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  11. Bimolecular affinity purification: a variation of TAP with multiple applications.

    PubMed

    Starokadomskyy, Petro; Burstein, Ezra

    2014-01-01

    The identification of true interacting partners of any given bait can be plagued by the nonspecific purification of irrelevant proteins. To avoid this problem, Tandem Affinity Purification (TAP) is a widely used procedure in molecular biology as this reduces the chance of nonspecific proteins being present in the final preparation. In this approach, two different affinity tags are fused to the protein bait. Herein, we review in detail a variation on the TAP procedure that we have previously developed, where the affinity moieties are placed on two different proteins that form a complex in vivo. This variation, which we refer to as Bimolecular Affinity Purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known proteins. We have utilized BAP for characterization of molecular complexes and evaluation of proteins interaction. Another application of BAP is the isolation of ubiquitin-like proteins (UBL)-modified fractions of a given protein and characterization of the lysine-acceptor site and structure of UBL-chains. PMID:24943324

  12. Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

    NASA Astrophysics Data System (ADS)

    Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

    2008-05-01

    Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

  13. Toward an Affinity Space Methodology: Considerations for Literacy Research

    ERIC Educational Resources Information Center

    Lammers, Jayne C.; Curwood, Jen Scott; Magnifico, Alecia Marie

    2012-01-01

    As researchers seek to make sense of young people's online literacy practices and participation, questions of methodology are important to consider. In our work to understand the culture of physical, virtual and blended spheres that adolescents inhabit, we find it necessary to expand Gee's (2004) notion of affinity spaces. In this article, we draw…

  14. Calculation of antibody affinity in homogeneous and heterogeneous systems.

    PubMed

    Chalquest, R R

    1988-12-01

    Antibody affinity is an important determinant of all antibody-antigen reactions. A new computer program, AFCRV, was developed to calculate binding constants with data from a radioimmunoassay on most microcomputers in the laboratory by using constant-ratio dilution curves. Evaluation of a homogeneous or heterogeneous antibody in the presence of a single antigen can be accomplished.

  15. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    PubMed Central

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6. PMID:27291418

  16. Valuing vaccination

    PubMed Central

    Bärnighausen, Till; Bloom, David E.; Cafiero-Fonseca, Elizabeth T.; O’Brien, Jennifer Carroll

    2014-01-01

    Vaccination has led to remarkable health gains over the last century. However, large coverage gaps remain, which will require significant financial resources and political will to address. In recent years, a compelling line of inquiry has established the economic benefits of health, at both the individual and aggregate levels. Most existing economic evaluations of particular health interventions fail to account for this new research, leading to potentially sizable undervaluation of those interventions. In line with this new research, we set forth a framework for conceptualizing the full benefits of vaccination, including avoided medical care costs, outcome-related productivity gains, behavior-related productivity gains, community health externalities, community economic externalities, and the value of risk reduction and pure health gains. We also review literature highlighting the magnitude of these sources of benefit for different vaccinations. Finally, we outline the steps that need to be taken to implement a broad-approach economic evaluation and discuss the implications of this work for research, policy, and resource allocation for vaccine development and delivery. PMID:25136129

  17. Univalent and Bivalent Ligands of Butorphan: Characteristics of the Linking Chain determine the Affinity and Potency of Such Opioid Ligands†

    PubMed Central

    Decker, Michael; Fulton, Brian S.; Zhang, Bin; Knapp, Brian I.; Bidlack, Jean M.; Neumeyer, John L.

    2009-01-01

    Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the μ, δ, and κ opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while only partially affecting binding affinity. The resulting bivalent ligands with optimized spacer-length and structure show potent binding profiles with the most potent compound (4b) having Ki values of 0.47 nM for both the μ and κ opioid receptors, and 4a having Ki values of 0.95 and 0.62 nM for the μ and κ receptors, respectively. Both 4a and 4b were partial agonists at the κ and μ receptors in the [35S]GTPγS binding assay. PMID:19634902

  18. Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand.

    PubMed

    Huang, Xinyi

    2003-02-01

    For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K(i) values that can be resolved is the K(d) value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.

  19. Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

    PubMed

    Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

    2014-11-01

    The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα.

  20. Novel trends in affinity biosensors: current challenges and perspectives

    NASA Astrophysics Data System (ADS)

    Arugula, Mary A.; Simonian, Aleksandr

    2014-03-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives.

  1. Myoglobin oxygen affinity in aquatic and terrestrial birds and mammals.

    PubMed

    Wright, Traver J; Davis, Randall W

    2015-07-01

    Myoglobin (Mb) is an oxygen binding protein found in vertebrate skeletal muscle, where it facilitates intracellular transport and storage of oxygen. This protein has evolved to suit unique physiological needs in the muscle of diving vertebrates that express Mb at much greater concentrations than their terrestrial counterparts. In this study, we characterized Mb oxygen affinity (P50) from 25 species of aquatic and terrestrial birds and mammals. Among diving species, we tested for correlations between Mb P50 and routine dive duration. Across all species examined, Mb P50 ranged from 2.40 to 4.85 mmHg. The mean P50 of Mb from terrestrial ungulates was 3.72±0.15 mmHg (range 3.70-3.74 mmHg). The P50 of cetaceans was similar to terrestrial ungulates ranging from 3.54 to 3.82 mmHg, with the exception of the melon-headed whale, which had a significantly higher P50 of 4.85 mmHg. Among pinnipeds, the P50 ranged from 3.23 to 3.81 mmHg and showed a trend for higher oxygen affinity in species with longer dive durations. Among diving birds, the P50 ranged from 2.40 to 3.36 mmHg and also showed a trend of higher affinities in species with longer dive durations. In pinnipeds and birds, low Mb P50 was associated with species whose muscles are metabolically active under hypoxic conditions associated with aerobic dives. Given the broad range of potential globin oxygen affinities, Mb P50 from diverse vertebrate species appears constrained within a relatively narrow range. High Mb oxygen affinity within this range may be adaptive for some vertebrates that make prolonged dives.

  2. Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance

    SciTech Connect

    Kuziemko, G.M.; Stroh, M.; Stevens, R.C. |

    1996-05-21

    The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 {times} 10{sup {minus}12} M for GM1 to 1.88 {times} 10{sup {minus}10} M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to K{sub d} values determined with whole-cell binding assays. Both whole-cell assays ans SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface. 34 refs., 8 figs., 2 tabs.

  3. Affinity of neuroleptics for D1 receptor of human brain striatum.

    PubMed Central

    Kanba, S; Suzuki, E; Nomura, S; Nakaki, T; Yagi, G; Asai, M; Richelson, E

    1994-01-01

    We determined the inhibition-dissociation constant (Ki) of a number of neuroleptics for D1 receptors of normal human brain tissue using [3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3[benzazepine-7- ol]. SCH23390 had the highest affinity with a Ki of 0.76 nM. Among clinically used drugs, propericiazine showed the highest affinity with a Ki of 10 nM. When neuroleptics were classified according to chemical structures, the Ki values were as follows. Phenothiazines ranged from 10 nM to 250 nM. Butyrophenones ranged from 45 nM to 250 nM. Thioxanthenes ranged from 12 nM to 340 nM. Orthopramines were more than 10,000 nM. The Ki values for the binding site of this study were significantly correlated with those reported in studies using animal brain. The possible relationship between D1 receptors and negative symptoms is discussed. PMID:7918347

  4. The electron affinities of C{sub 3}O and C{sub 4}O

    SciTech Connect

    Rienstra-Kiracofe, J.C.; Ellison, G.B.; Hoffman, B.C.; Schaefer, H.F. III

    2000-03-23

    The authors predict the adiabatic electron affinities of C{sub 3}O and C{sub 4}O based on electronic structure calculations, using a large triple-{zeta} basis set with polarization and diffuse functions (TZ2Pf+diff) with the SCF, CCSD, and CCSD(T) methods as well as with the aug-cc-pVDZ and aug-cc-pVTZ basis sets. The results imply electron affinities for C{sub 3}O and C{sub 4}O; EA(C{sub 3}O) = 0.93 eV {+-} 0.10 and EA(C{sub 4}O) = 2.99 {+-} 0.10. The EA(C{sub 3}O) is 0.41 eV lower than the experimental value of 1.34 {+-} 0.15 eV, while the EA(C{sub 4}O) is 0.94 eV higher than the experimental value of 2.05 {+-} 0.15 eV. Optimized geometries for all species at each level of theory are given, and harmonic vibrational frequencies are reported at the SCF/TZ2Pf+diff and CCSD/aug-cc-pVDZ levels.

  5. Phosphate-affinity electrophoresis on a microchip for determination of protein kinase activity.

    PubMed

    Han, Aishan; Hosokawa, Kazuo; Maeda, Mizuo

    2009-10-01

    We describe microchip-based phosphate-affinity electrophoresis (microPAE) for separation of peptides aimed at determination of kinase activity. The microPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form. We synthesized a Phos-tag-poly(dimethylacrylamide) conjugate. The conjugate and the sample solutions were autonomously injected into a PDMS-glass hybrid microchip. The two solutions were contacted together in the microchannel. When the peptides were electrophoresed into the Phos-tag-poly(dimethylacrylamide) region, the phosphorylated peptide was specifically trapped, and separated from the nonphosphorylated peptide in 10 s. The results were quantified by the areas of the fluorescence peaks. The calibration plot obtained with standard samples showed an excellent linearity and a LOD of 0.9% phosphorylated peptide among the total peptides. For c-Src-reacted samples, the results from the microPAE were in good agreement with those from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The microPAE was also successful in the presence of inhibitors for c-Src. The measured 50% inhibitory concentration values for staurosporine, PP2, and SU6656 were in good agreement with the literature values. PMID:19784951

  6. Non-affine fields in solid-solid transformations: the structure and stability of a product droplet.

    PubMed

    Paul, Arya; Sengupta, Surajit; Rao, Madan

    2014-01-01

    We describe the microstructure, morphology, and dynamics of growth of a droplet of martensite nucleating in a parent austenite during a solid-solid transformation, using a Landau theory written in terms of both conventional affine elastic deformations and non-affine deformations. Non-affineness, φ, serves as a source of strain incompatibility and screens long-ranged elastic interactions. It is produced wherever the local stress exceeds a threshold and anneals diffusively thereafter. Using a variational calculation, we find three types of stable solution (labeled I, II, and III) for the structure of the product droplet, depending on the stress threshold and the scaled mobilities of φ parallel and perpendicular to the parent-product interface. The profile of the non-affine field φ is different in these three solutions: I is characterized by a vanishingly small φ, II admits large values of φ localized in regions of high stress within the parent-product interface, and III is a structure in which φ completely wets the parent-product interface. The width l and size W of the twins follow the relation l is proportional to √W in solution I; this relation does not hold for II or III. We obtain a dynamical phase diagram featuring these solutions, and argue that they represent specific solid-state microstructures.

  7. Density Functional Study of Structures and Electron Affinities of BrO4F/BrO4F−

    PubMed Central

    Gong, Liangfa; Xiong, Jieming; Wu, Xinmin; Qi, Chuansong; Li, Wei; Guo, Wenli

    2009-01-01

    The structures, electron affinities and bond dissociation energies of BrO4F/BrO4F− species have been investigated with five density functional theory (DFT) methods with DZP++ basis sets. The planar F-Br…O2…O2 complexes possess 3A′ electronic state for neutral molecule and 4A′ state for the corresponding anion. Three types of the neutral-anion energy separations are the adiabatic electron affinity (EAad), the vertical electron affinity (EAvert), and the vertical detachment energy (VDE). The EAad value predicted by B3LYP method is 4.52 eV. The bond dissociation energies De (BrO4F → BrO4-mF + Om) (m = 1–4) and De− (BrO4F− → BrO4-mF− + Om and BrO4F− → BrO4-mF + Om−) are predicted. The adiabatic electron affinities (EAad) were predicted to be 4.52 eV for F-Br…O2…O2 (3A′←4A′) (B3LYP method). PMID:19742128

  8. Design of Bcl-2 and Bcl-xL Inhibitors with Subnanomolar Binding Affinities Based upon a New Scaffold

    SciTech Connect

    Zhou, Haibin; Chen, Jianfang; Meagher, Jennifer L.; Yang, Chao-Yie; Aguilar, Angelo; Liu, Liu; Bai, Longchuan; Cong, Xin; Cai, Qian; Fang, Xueliang; Stuckey, Jeanne A.; Wang, Shaomeng

    2014-10-02

    Employing a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of the anti-apoptotic proteins Bcl-2 and Bcl-xL. An initial lead compound with a new scaffold was designed based upon the crystal structure of Bcl-xL and U.S. Food and Drug Administration (FDA) approved drugs and was found to have an affinity of 100 {micro}M for both Bcl-2 and Bcl-xL. Linking this weak lead to another weak-affinity fragment derived from Abbott's ABT-737 led to an improvement of the binding affinity by a factor of >10,000. Further optimization ultimately yielded compounds with subnanomolar binding affinities for both Bcl-2 and Bcl-xL and potent cellular activity. The best compound (21) binds to Bcl-xL and Bcl-2 with K{sub i} < 1 nM, inhibits cell growth in the H146 and H1417 small-cell lung cancer cell lines with IC{sub 50} values of 60-90 nM, and induces robust cell death in the H146 cancer cell line at 30-100 nM.

  9. Positron-attachment to small molecules: Vibrational enhancement of positron affinities with configuration interaction level of multi-component molecular orbital approach

    SciTech Connect

    Tachikawa, Masanori

    2015-12-31

    To theoretically demonstrate the binding of a positron to small polarized molecules, we have calculated the vibrational averaged positron affinity (PA) values along the local vibrational contribution with the configuration interaction level of multi-component molecular orbital method. This method can take the electron-positron correlation contribution into account through single electronic - single positronic excitation configurations. The PA values are enhanced by including the local vibrational contribution from vertical PA values due to the anharmonicity of the potential.

  10. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  11. Proton affinities and photoelectron spectra of phenylalanine and N-methyl- and N,N-dimethylphenylalanine. Correlation of lone pair ionization energies with proton affinities and implications for N-methylation as a method to effect site specific protonation of peptides

    SciTech Connect

    Campbell, S.; Marzluff, E.M.; Rodgers, M.T.; Beauchamp, J.L. ); Rempe, M.E.; Schwinck, K.F.; Lichtenberger, D.L. )

    1994-06-15

    A Fourier transform ion cyclotron resonance (FT-ICR) technique for measuring gas-phase proton affinities is presented which utilizes collisional dissociation of proton-bound clusters by off-resonance translational excitation. A simplified RRKM analysis relates unimolecular dissociation rates to proton affinities. This technique is used to measure values for the proton affinities of phenylalanine and N-methyl- and N,N-dimethylphenylalanine of 220.3, 223.6, and 224.5 kcal/mol, respectively (relative to the proton affinity of NH[sub 3] = 204.0 kcal/mol). The proton affinity measured for phenylalanine is in excellent agreement with reported literature values. The photoelectron spectra of these three molecules are also presented and analyzed. Assignments of bands to specific ionization processes are aided by comparison with model compounds such as methyl-substituted amines and 2-phenylethylamines. These data are employed to examine the correlation of adiabatic nitrogen lone pair ionization energies with gas-phase proton affinities for phenylalanine, N-methylphenylalanine, and N,N-dimethylphenylalanine in comparison to correlations for other amino acids and selected aliphatic amines. 41 refs., 7 figs., 2 tabs.

  12. F+ and F⁻ affinities of simple N(x)F(y) and O(x)F(y) compounds.

    PubMed

    Grant, Daniel J; Wang, Tsang-Hsiu; Vasiliu, Monica; Dixon, David A; Christe, Karl O

    2011-03-01

    Atomization energies at 0 K and heats of formation at 0 and 298 K are predicted for the neutral and ionic N(x)F(y) and O(x)F(y) systems using coupled cluster theory with single and double excitations and including a perturbative triples correction (CCSD(T)) method with correlation consistent basis sets extrapolated to the complete basis set (CBS) limit. To achieve near chemical accuracy (±1 kcal/mol), three corrections to the electronic energy were added to the frozen core CCSD(T)/CBS binding energies: corrections for core-valence, scalar relativistic, and first order atomic spin-orbit effects. Vibrational zero point energies were computed at the CCSD(T) level of theory where possible. The calculated heats of formation are in good agreement with the available experimental values, except for FOOF because of the neglect of higher order correlation corrections. The F(+) affinity in the N(x)F(y) series increases from N(2) to N(2)F(4) by 63 kcal/mol, while that in the O(2)F(y) series decreases by 18 kcal/mol from O(2) to O(2)F(2). Neither N(2) nor N(2)F(4) is predicted to bind F(-), and N(2)F(2) is a very weak Lewis acid with an F(-) affinity of about 10 kcal/mol for either the cis or trans isomer. The low F(-) affinities of the nitrogen fluorides explain why, in spite of the fact that many stable nitrogen fluoride cations are known, no nitrogen fluoride anions have been isolated so far. For example, the F(-) affinity of NF is predicted to be only 12.5 kcal/mol which explains the numerous experimental failures to prepare NF(2)(-) salts from the well-known strong acid HNF(2). The F(-) affinity of O(2) is predicted to have a small positive value and increases for O(2)F(2) by 23 kcal/mol, indicating that the O(2)F(3)(-) anion might be marginally stable at subambient temperatures. The calculated adiabatic ionization potentials and electron affinities are in good agreement with experiment considering that many of the experimental values are for vertical processes.

  13. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323).

  14. Canary islands-north African population affinities: measures of divergence based on dental morphology.

    PubMed

    Guatelli-Steinberg, D; Irish, J D; Lukacs, J R

    2001-01-01

    This investigation addresses two related questions about the origins and biological affinities of the Canary Islands' aboriginal inhabitants. First: With which North African populations do the pre-conquest inhabitants of the Canary Islands have their greatest affinities? Second: Does inter-island biological variability among the Canary Islanders, as has been suggested by other researchers (Hooton 1925, Schwidetzky 1963), imply that potentially different founding populations remained distinct during the pre-conquest period? This study employs dental morphology data derived from pre-conquest skeletons to answer these questions. Non-metric dental traits appear to be controlled by polygenic systems with a low to moderate environmental contribution to the resulting phenotype (Berry 1978, Harris & Bailit 1980, Nichol 1990) and can thus be assumed to reflect genetic relationships. The dental morphology of a sample of Canary Islanders (n = 397) is compared to that of Northwest African samples of Algerian Shawia Berbers (n = 26), Kabyle Berbers (n = 32), Bedouin Arabs (n = 49) and Punic Carthaginians (n = 28) as well as to six samples from Northeast Africa (n = 307) included for the purpose of understanding Canary Islanders' affinities within a wider context. The analysis employs 28 dental traits, quantifying differences in their expression among the various samples through a summary statistic, CAB Smith's Mean Measure of Divergence (MMD). The MMD analysis indicates that the Canary Island sample is most similar to the four samples from Northwest Africa: the Shawia Berbers, Kabyle Berbers, Bedouin Arabs and Carthaginians, less similar to the three Egyptian samples and least like the three Nubian samples. An intra-island comparison among samples from La Gomera, Gran Canaria and Tenerife reveals low, insignificant MMD values in all cases, implying that inter-island dental morphology differences are not so great as to require hypotheses of separate founding populations.

  15. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323). PMID:25757499

  16. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    SciTech Connect

    Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

    1980-10-10

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo(/sup 14/C/sub 2/)acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

  17. Histology and affinity of the earliest armoured vertebrate.

    PubMed

    Sansom, Ivan J; Donoghue, Philip C J; Albanesi, Guillermo

    2005-12-22

    Arandaspids are the earliest skeletonizing vertebrates known from articulated remains. Despite a wealth of data, their affinity remains questionable because they exhibit a random mixture of primitive and derived characteristics. We constrain the affinity of arandaspids by providing the first detailed characterization of their dermoskeleton which is revealed to be three-layered, composed of a basal laminated, cancellous middle and tubercular superficial layers. All three layers are composed of acellular bone but the superficial layer also includes dentine and enameloid, comprising the tubercles. As such, the composition of the arandaspid dermoskeleton is common to heterostracans and astraspids, supporting existing hypotheses of early vertebrate phylogeny. This emphasizes the peculiarity of existing interpretations of aranadaspid anatomy and there is need for a complete reappraisal of the existing anatomical data.

  18. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  19. Evolution based on chromosome affinity from a network perspective

    NASA Astrophysics Data System (ADS)

    Monteiro, R. L. S.; Fontoura, J. R. A.; Carneiro, T. K. G.; Moret, M. A.; Pereira, H. B. B.

    2014-06-01

    Recent studies have focused on models to simulate the complex phenomenon of evolution of species. Several studies have been performed with theoretical models based on Darwin's theories to associate them with the actual evolution of species. However, none of the existing models include the affinity between individuals using network properties. In this paper, we present a new model based on the concept of affinity. The model is used to simulate the evolution of species in an ecosystem composed of individuals and their relationships. We propose an evolutive algorithm that incorporates the degree centrality and efficiency network properties to perform the crossover process and to obtain the network topology objective, respectively. Using a real network as a starting point, we simulate its evolution and compare its results with the results of 5788 computer-generated networks.

  20. Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands.

    PubMed

    Kim, Yaewon; Hilty, Christian

    2015-04-13

    Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.

  1. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  2. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

  3. Affinity Propagation Clustering of Measurements for Multiple Extended Target Tracking

    PubMed Central

    Zhang, Tao; Wu, Renbiao

    2015-01-01

    More measurements are generated by the target per observation interval, when the target is detected by a high resolution sensor, or there are more measurement sources on the target surface. Such a target is referred to as an extended target. The probability hypothesis density filter is considered an efficient method for tracking multiple extended targets. However, the crucial problem of how to accurately and effectively partition the measurements of multiple extended targets remains unsolved. In this paper, affinity propagation clustering is introduced into measurement partitioning for extended target tracking, and the elliptical gating technique is used to remove the clutter measurements, which makes the affinity propagation clustering capable of partitioning the measurement in a densely cluttered environment with high accuracy. The Gaussian mixture probability hypothesis density filter is implemented for multiple extended target tracking. Numerical results are presented to demonstrate the performance of the proposed algorithm, which provides improved performance, while obviously reducing the computational complexity. PMID:26370998

  4. Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses.

    PubMed

    Eisen, Herman N

    2014-05-01

    The antibodies produced initially in response to most antigens are high molecular weight (MW) immunoglobulins (IgM) with low affinity for the antigen, while the antibodies produced later are lower MW classes (e.g., IgG and IgA) with, on average, orders of magnitude higher affinity for that antigen. These changes, often termed affinity maturation, take place largely in small B-cell clusters (germinal center; GC) in lymphoid tissues in which proliferating antigen-stimulated B cells express the highly mutagenic cytidine deaminase that mediates immunoglobulin class-switching and sequence diversification of the immunoglobulin variable domains of antigen-binding receptors on B cells (BCR). Of the large library of BCR-mutated B cells thus rapidly generated, a small minority with affinity-enhancing mutations are selected to survive and differentiate into long-lived antibody-secreting plasma cells and memory B cells. BCRs are also endocytic receptors; they internalize and cleave BCR-bound antigen, yielding peptide-MHC complexes that are recognized by follicular helper T cells. Imperfect correlation between BCR affinity for antigen and cognate T-cell engagement may account for the increasing affinity heterogeneity that accompanies the increasing average affinity of antibodies. Conservation of mechanisms underlying mutation and selection of high-affinity antibodies over the ≈200 million years of evolution separating bird and mammal lineages points to the crucial role of antibody affinity enhancement in adaptive immunity.

  5. High affinity DNA-microtubule associated protein interaction.

    PubMed

    Marx, K A

    1992-07-01

    We have isolated the MAP/tau proteins from twice-cycled chick brain microtubule preparations and demonstrated that they are responsible for the nitrocellulose DNA binding activity we and others have measured. Using the isolated MAP/tau proteins we then measured the apparent affinity constant K(app) for the homologous chick DNA interaction and found evidence for two equilibrium affinity classes-a K(app) = 6 x 10(7) M-1, responsible for the bulk of the DNA binding activity and a small (less than 10%) higher affinity K(app) = 10(8) - 10(9) M-1, likely due to sequence specific binding protein species. Using the same chick brain MAP-tau protein, a heterologous interaction with D. melanogaster DNA, was found to possess just the lower affinity class-K(app) = 2 x 10(7) M-1. Under stringent binding conditions we carried out equilibrium nitrocellulose filter binding experiments in a ternary reaction mixture at constant MAP/tau protein and 35S radiolabelled chick DNA concentration using increasing and excess concentrations of competitor DNAs of different sources. The order of competitor strengths found was-chick DNA greater than mouse DNA greater than D. melanogaster = E. coli. DNA. These data and specifically the homologous DNA: protein case being the strongest competitor corroborate our previous studies using total microtubule protein and provide new evidence for a conserved interaction of a small DNA sequence class with MAP/tau protein species. Moreover, these data allow us to conclude that the conserved DNA sequence: MAP/tau protein interactions do not critically depend upon any energetic feature co-involving tubulin for their properties since tubulin is absent from these preparations.

  6. Chlorophenylpiperazine analogues as high affinity dopamine transporter ligands.

    PubMed

    Motel, William C; Healy, Jason R; Viard, Eddy; Pouw, Buddy; Martin, Kelly E; Matsumoto, Rae R; Coop, Andrew

    2013-12-15

    Selective σ2 ligands continue to be an active target for medications to attenuate the effects of psychostimulants. In the course of our studies to determine the optimal substituents in the σ2-selective phenyl piperazines analogues with reduced activity at other neurotransmitter systems, we discovered that 1-(3-chlorophenyl)-4-phenethylpiperazine actually had preferentially increased affinity for dopamine transporters (DAT), yielding a highly selective DAT ligand. PMID:24211020

  7. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  8. Selective high affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  9. pp-waves with torsion and metric-affine gravity

    NASA Astrophysics Data System (ADS)

    Pasic, Vedad; Vassiliev, Dmitri

    2005-10-01

    A classical pp-wave is a four-dimensional Lorentzian spacetime which admits a nonvanishing parallel spinor field; here the connection is assumed to be Levi-Civita. We generalize this definition to metric compatible spacetimes with torsion and describe basic properties of such spacetimes. We use our generalized pp-waves for constructing new explicit vacuum solutions of quadratic metric-affine gravity.

  10. Affine.m—Mathematica package for computations in representation theory of finite-dimensional and affine Lie algebras

    NASA Astrophysics Data System (ADS)

    Nazarov, Anton

    2012-11-01

    In this paper we present Affine.m-a program for computations in representation theory of finite-dimensional and affine Lie algebras and describe implemented algorithms. The algorithms are based on the properties of weights and Weyl symmetry. Computation of weight multiplicities in irreducible and Verma modules, branching of representations and tensor product decomposition are the most important problems for us. These problems have numerous applications in physics and we provide some examples of these applications. The program is implemented in the popular computer algebra system Mathematica and works with finite-dimensional and affine Lie algebras. Catalogue identifier: AENA_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENB_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, UK Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 24 844 No. of bytes in distributed program, including test data, etc.: 1 045 908 Distribution format: tar.gz Programming language: Mathematica. Computer: i386-i686, x86_64. Operating system: Linux, Windows, Mac OS, Solaris. RAM: 5-500 Mb Classification: 4.2, 5. Nature of problem: Representation theory of finite-dimensional Lie algebras has many applications in different branches of physics, including elementary particle physics, molecular physics, nuclear physics. Representations of affine Lie algebras appear in string theories and two-dimensional conformal field theory used for the description of critical phenomena in two-dimensional systems. Also Lie symmetries play a major role in a study of quantum integrable systems. Solution method: We work with weights and roots of finite-dimensional and affine Lie algebras and use Weyl symmetry extensively. Central problems which are the computations of weight multiplicities, branching and fusion coefficients are solved using one general recurrent

  11. MH-3: evidence for non-competitive antagonism towards the low-affinity site of β1-adrenoceptors.

    PubMed

    Schlicker, Eberhard; Pędzińska-Betiuk, Anna; Kozłowska, Hanna; Szkaradek, Natalia; Żelaszczyk, Dorota; Baranowska-Kuczko, Marta; Kieć-Kononowicz, Katarzyna; Marona, Henryk; Malinowska, Barbara

    2014-08-01

    β-Adrenoceptor antagonists are important drugs for the treatment of cardiovascular diseases and some of those drugs also block the so-called low-affinity site of β1-adrenoceptors although at much higher concentrations. This low-affinity site, also identified in vivo and in human tissue, may come into play under certain pathophysiological situations including arrhythmias. The aim of our study was to determine the potency of 14 compounds chemically related to bupranolol or bevantolol and two xanthone derivatives at the low-affinity site of the β1-adrenoceptor. The potency of the compounds at the low- and high-affinity site of β1-adrenoceptors (β1L and β1H; both increasing heart rate) was compared in the pithed rat. One compound was also studied in the isolated rat heart and its α1-adrenolytic effect determined in the isolated rat mesenteric artery. In the pithed rat, four compounds blocked the β1L-adrenoceptor at a ≥10-fold lower potency than the β1H-adrenoceptor whereas the xanthone derivative (-)-MH-3 was equipotent. In the spontaneously beating right atrium (-)-MH-3 was a non-competitive antagonist of comparable potency at either receptor; its apparent pD'2 value for the β1L-adrenoceptor ranged from 5.6 to 6.4 under various conditions, including the Langendorff preparation. Its apparent pA2 at the α1-adrenoceptor in the mesenteric artery was 8.4. (-)-MH-3 is the first compound with virtually the same potency at the low- and high-affinity site of β1-adrenoceptors in vivo; it appears to be a non-competitive antagonist at either site in vitro.

  12. When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

    NASA Astrophysics Data System (ADS)

    Petre, Brînduşa-Alina; Ulrich, Martina; Stumbaum, Mihaela; Bernevic, Bogdan; Moise, Adrian; Döring, Gerd; Przybylski, Michael

    2012-11-01

    Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

  13. Metal-polybenzimidazole complexes as a nonviral gene carrier: effects of the DNA affinity on gene delivery.

    PubMed

    Huang, Xueying; Dong, Xiongwei; Li, Xue; Meng, Xianggao; Zhang, Dan; Liu, Changlin

    2013-12-01

    The metal complex-based carriers are emerging likely as a new type of gene-delivery systems prone to systematic structural alteration and chemical tailoring. In our work, the DNA affinity of metal complexes with polybenzimidazoles was found to be one of the determinants that can regulate expression of the transgenes. Here, the correlations between the DNA affinity and transfection efficacy were explored by characterizing gene-delivering properties of a series of Co(2+)- and Ca(2+)-polybenzimidazole complexes. The binding equilibrium constants (Kobs) of the divalent metal complexes to DNA, which is considered as a measure of the DNA affinity of metal complexes, were evaluated by isothermal titration calorimetry (ITC) and UV-visible absorption titration. The properties of DNA condensates formed with the metal complexes including sizes, ζ potential and morphology were observed to be altered with Kobs values. The monodispersed spherical condensates were found only for the Ca(2+) complexes whose DNA affinity is weaker than that of the Co(2+) complexes. However, the cell internalization examination indicated that cell uptake of the DNA condensates is independent of homogeneity in their sizes and morphology. The comparison of transgene expression showed that that the Ca(2+) complex-mediated transfection has higher efficiency than the Co(2+) complexes under the conditions tested, and the transfection efficacy cannot be correlated with the cell uptake of DNA condensates. Moreover, the Ca(2+) complexes and their DNA condensates had lower cytotoxicity than the Co(2+) complexes. Thus, the DNA affinity should be one of the factors to be capable of regulating the gene-delivering property of metal complexes. PMID:24099694

  14. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    PubMed

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  15. Nanoparticle Surface Affinity as a Predictor of Trophic Transfer.

    PubMed

    Geitner, Nicholas K; Marinakos, Stella M; Guo, Charles; O'Brien, Niall; Wiesner, Mark R

    2016-07-01

    Nanoscale materials, whether natural, engineered, or incidental, are increasingly acknowledged as important components in large, environmental systems with potential implications for environmental impact and human health. Mathematical models are a useful tool for handling the rapidly increasing complexity and diversity of these materials and their exposure routes. Presented here is a mathematical model of trophic transfer driven by nanomaterial surface affinity for environmental and biological surfaces, developed in tandem with an experimental functional assay for determining these surface affinities. We found that nanoparticle surface affinity is a strong predictor of uptake through predation in a simple food web consisting of the algae Chlorella vulgaris and daphnid Daphnia magna. The mass of nanoparticles internalized by D. magna through consuming nanomaterial-contaminated algae varied linearly with surface-attachment efficiency. Internalized quantities of gold nanoparticles in D. magna ranged from 8.3 to 23.6 ng/mg for nanoparticle preparations with surface-attachment efficiencies ranging from 0.07 to 1. This model, coupled with the functional-assay approach, may provide a useful screening tool for existing materials as well as a predictive model for their development. PMID:27249534

  16. Affine transformations capture beak shape variation in Darwin's Finches

    NASA Astrophysics Data System (ADS)

    Brenner, Michael; Campas, Otger; Mallarino, Riccardo; Abzhanov, Arhat

    2009-11-01

    Evolution by natural selection has resulted in extraordinary morphological complexity of living organisms, whose description has thus far defied any precise mathematical characterization linked to the underlying developmental genetics. Here we demonstrate that the morphological diversity of the beaks of Darwin's finches, the classical example of adaptive morphological radiation, is quantitatively accounted for through the mathematical group of affine transformations. Specifically, we show that all beak shapes of Ground Finches (genus Geospiza) are related by scaling transformations (a subgroup of the affine group), and the same scheme occurs for all the beak shapes of Tree and Warbler finches. This analysis shows that the beak shapes within each of these groups differ only by their scales, such as length and depth, each of which is knownto be under genetic control.The complete morphological variability within the beaks of Darwin's finches can be explained by extending the scaling transformations to the entire affine group, by including shear transformations. Altogether our results suggest that the mathematical theory of groups can help decode morphological variability, and points to a potentially hierarchical structure of morphological diversity and the underlying developmental processes.

  17. Single-cell measurement of red blood cell oxygen affinity

    PubMed Central

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

  18. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  19. Comparison of Inlet Geometry in Microfluidic Cell Affinity Chromatography

    PubMed Central

    Li, Peng; Tian, Yu; Pappas, Dimitri

    2011-01-01

    Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design has not been extensively described or studied. The most common inlets—used to connect the microfluidic chip to pumps, tubing, etc—are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performance of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than the remaining channel, while for parallel inlet chips cell density at the inlet area was similar to the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices. PMID:21207967

  20. Negative Enrichment of Target Cells by Microfluidic Affinity Chromatography

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2011-01-01

    A three-dimensional microfluidic channel was developed for high purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of non-target cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled, and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multi-step processes in microfluidic systems. In previous work, we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity, ease of fabrication and use, is suitable for cell separations when subsequent analysis of target cells is required. PMID:21939198

  1. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    PubMed

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-01

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166

  2. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Graça, Vânia C; Sousa, Fani; Santos, Paulo F; Almeida, Paulo S

    2015-01-01

    Affinity chromatography (AC) is one of the most important techniques for the separation and purification of biomolecules, being probably the most selective technique for protein purification. It is based on unique specific reversible interactions between the target molecule and a ligand. In this affinity interaction, the choice of the ligand is extremely important for the success of the purification protocol. The growing interest in AC has motivated an intense research effort toward the development of materials able to overcome the disadvantages of conventional natural ligands, namely their high cost and chemical and biological lability. In this context, synthetic dyes have emerged, in recent decades, as a promising alternative to biological ligands. Herein, detailed protocols for the assembling of a new chromatographic dye-ligand affinity support bearing an immobilized aminosquarylium cyanine dye on an agarose-based matrix (Sepharose CL-6B) and for the separation of a mixture o f three standard proteins: lysozyme, α-chymotrypsin, and trypsin are provided. PMID:25749942

  3. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. PMID:26830537

  4. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

  5. Affinity labeling of the ribosomal P site in Drosophila melanogaster

    SciTech Connect

    North, D.

    1987-01-01

    Several recent studies have probed the peptidyl transferase region of the Drosophila ribosome via the use of reactive site specific analogues (affinity labels). P site proteins adjacent to the 3' end of the amino acid bearing tRNA strand were labeled with modified tRNA fragments. Drugs affecting the binding of these agents were used to further clarify the nature of the region. The nascent peptide region of the P site was not labeled in previous experiments. To label that region radioactive Bromoacetylphenylalanyl-tRNA (BrAcphe-tRNA) was synthesized. The alpha-bromoacetyl group of this analogue is potentially reactive with nucleophiles present in either proteins or RNAs. Charged tRNAs and tRNA analogues bearing a peptide bond on the N-terminus of their amino acid are recognized as having affinity for the ribosomal P site. Specific labeling of the P site by BrAcphe-tRNA was confirmed by its ability to radioactively label proteins indirectly. As many as 8 ribosomal proteins may be labeled under these conditions, however, the majority of the bound label is associated with 3 large subunit proteins and 2 small subunit proteins. Overlaps between the proteins labeled by BrAcphe-tRNA and those labeled by other affinity labels are examined and a model of the peptidyl transferase region of Drosophila ribosomes is presented.

  6. Multiple lectin detection by cell membrane affinity binding.

    PubMed

    Ribeiro, Ana; Catarino, Sofia; Ferreira, Ricardo Boavida

    2012-05-01

    Assuming that lectins evolved to recognise relatively complex and branched oligosaccharides or parts of them, rather than simple sugars, a procedure based on lectin affinity binding to isolated erythrocyte (or any other cell type) membranes is proposed. This methodology was validated using six pure commercial lectins, as well as lectins from total protein extracts of Arbutus unedo leaves. All commercial lectins, as well as five polypeptides from A. unedo leaves bound to the glycosylated membrane receptors and were eluted by the corresponding sugars. When compared to the standard affinity chromatography procedure involving an individual sugar bound to a solid matrix, the new method provides a single-step, effective detection method for lectins and allows the rapid screening of their profile present in any unknown protein solution, indicates their biological carbohydrate affinities as well as their sugar specificities (if any), enables the simultaneous analysis of a large number of samples, does not require any pre-purification steps, permits detection of additional lectins and provides data which are more relevant from the physiological point of view. PMID:22381939

  7. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  8. Relative binding affinities of monolignols to horseradish peroxidase

    DOE PAGES

    Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C.

    2016-07-22

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic –OH group andmore » a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic –OH group instead interacting with Pro139. Furthermore, since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.« less

  9. Molecular fine-tuning of affinity maturation in germinal centers.

    PubMed

    Wu, Yee Ling; Rada, Cristina

    2016-01-01

    The development of high-affinity antibodies in response to infection is an iterative process in which B cells cycle between proliferation/somatic hypermutation and antigen-driven selection. These processes occur within specific regions of the secondary lymphoid structures known as germinal centers (GCs) and the environmental and signaling cues provided by these regions guide the GC reactions that drive B cell maturation and antibody production, ultimately determining B cell fate. In this issue of the JCI, Nakagawa and colleagues examine the role of miR-155, a microRNA that is required for GC development and the production of high-affinity antibodies. They show that miR-155 is highly expressed in positively selected B cells and promotes survival of these cells by orienting the Myc transcription program toward survival rather than apoptosis through the inhibition of the transcriptional regulator JARID2. These findings illustrate the fine balance between apoptosis and proliferation that is required for the development of high-affinity antibodies.

  10. Relative Binding Affinities of Monolignols to Horseradish Peroxidase.

    PubMed

    Sangha, Amandeep K; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C

    2016-08-11

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic -OH group and a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic -OH group instead interacting with Pro139. Since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate. PMID:27447548

  11. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  12. Gas-phase lithium cation affinity of glycine.

    PubMed

    Bourcier, Sophie; Chiaa, Ru Xuan; Mimbong, Rosa Ngo Biboum; Bouchoux, Guy

    2015-01-01

    The gas-phase lithium cation binding thermochemistry of glycine has been determined theoretically by quantum chemical calculations at the G4 level and experimentally by the extended kinetic method using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. The lithium cation affinity of glycine, ∆(Li)H°(298)(GLY), i.e. the∆(Li)H°(298) of the reaction GlyLi(+)→ Gly + Li(+)) given by the G4 method is equal to 241.4 kJ.mol(-1) if only the most stable conformer of glycine is considered or to 242.3 kJ.mol(-1) if the 298K equilibrium mixture of neutral conformers is included in the calculation. The ∆(Li)H°(298)(GLY) deduced from the extended kinetic method is obviously dependent on the choice of the Li(+) affinity scale, thus∆(Li)H°(298)(GLY) is equal to 228.7±0.9(2.0) kJ.mol(- 1) if anchored to the recently re-evaluated lithium cation affinity scale but shifted to 235.4±1.0 kJ.mol(-1) if G4 computed lithium cation affinities of the reference molecules is used. This difference of 6.3 kJ.mol(-1) may originate from a compression of the experimental lithium affinity scale in the high ∆(Li)H°(298) region. The entropy change associated with the reaction GlyLi(+)→Gly + Li(+) reveals a gain of approximately 15 J.mol(-) 1.K(-1) with respect to monodentate Li(+) acceptors. The origin of this excess entropy is attributed to the bidentate interaction between the Li(+) cation and both the carbonyl oxygen and the nitrogen atoms of glycine. The computed G4 Gibbs free energy,∆(Li)G°(298)(GLY) is equal to 205.3 kJ.mol(-1), a similar result, 201.0±3.4 kJ.mol(-1), is obtained from the experiment if the∆(Li)G°(298) of the reference molecules is anchored on the G4 results. PMID:26307695

  13. Gas-phase lithium cation affinity of glycine.

    PubMed

    Bourcier, Sophie; Chiaa, Ru Xuan; Mimbong, Rosa Ngo Biboum; Bouchoux, Guy

    2015-01-01

    The gas-phase lithium cation binding thermochemistry of glycine has been determined theoretically by quantum chemical calculations at the G4 level and experimentally by the extended kinetic method using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. The lithium cation affinity of glycine, ∆(Li)H°(298)(GLY), i.e. the∆(Li)H°(298) of the reaction GlyLi(+)→ Gly + Li(+)) given by the G4 method is equal to 241.4 kJ.mol(-1) if only the most stable conformer of glycine is considered or to 242.3 kJ.mol(-1) if the 298K equilibrium mixture of neutral conformers is included in the calculation. The ∆(Li)H°(298)(GLY) deduced from the extended kinetic method is obviously dependent on the choice of the Li(+) affinity scale, thus∆(Li)H°(298)(GLY) is equal to 228.7±0.9(2.0) kJ.mol(- 1) if anchored to the recently re-evaluated lithium cation affinity scale but shifted to 235.4±1.0 kJ.mol(-1) if G4 computed lithium cation affinities of the reference molecules is used. This difference of 6.3 kJ.mol(-1) may originate from a compression of the experimental lithium affinity scale in the high ∆(Li)H°(298) region. The entropy change associated with the reaction GlyLi(+)→Gly + Li(+) reveals a gain of approximately 15 J.mol(-) 1.K(-1) with respect to monodentate Li(+) acceptors. The origin of this excess entropy is attributed to the bidentate interaction between the Li(+) cation and both the carbonyl oxygen and the nitrogen atoms of glycine. The computed G4 Gibbs free energy,∆(Li)G°(298)(GLY) is equal to 205.3 kJ.mol(-1), a similar result, 201.0±3.4 kJ.mol(-1), is obtained from the experiment if the∆(Li)G°(298) of the reference molecules is anchored on the G4 results.

  14. Robust adaptive control for a class of uncertain non-affine nonlinear systems using affine-type neural networks

    NASA Astrophysics Data System (ADS)

    Zhao, Shitie; Gao, Xianwen

    2016-08-01

    A robust adaptive control is proposed for a class of single-input single-output non-affine nonlinear systems. In order to approximate the unknown nonlinear function, a novel affine-type neural network is used, and then to compensate the approximation error and external disturbance a robust control term is employed. By Lyapunov stability analysis for the closed-loop system, it is proved that tracking errors asymptotically converge to zero. Moreover, an observer is designed to estimate the system states because all the states may not be available for measurements. Furthermore, the adaptation laws of neural networks and the robust controller are given out based on the Lyapunov stability theory. Finally, two simulation examples are presented to demonstrate the effectiveness of the proposed control method.

  15. Heparin affin regulatory peptide modulates the endogenous anticoagulant activity of heparin and heparan sulphate mimetics.

    PubMed

    Mejdoubi-Charef, Najet; Courty, José; Sineriz, Fernando; Papy-Garcia, Dulce; Charef, Said

    2012-11-01

    Pleiotrophin, also known as heparin affin regulatory peptide (HARP), is a growth factor expressed in various tissues and cell lines. In this work, HARP was tested for its capacity to modulate the anticoagulant activity of heparin and heparan sulphate mimetics (OTR4120). We used both in vitro and in vivo assays. HARP was found to be differently effective for neutralization of the anticoagulant activity of the mimetic heparan sulphate (OTR4120) and heparin in purified system and human plasma. HARP was shown to compete with both antithrombin and thrombin for binding to heparin and to OTR4120, respectively. In the presence of OTR4120, the V(max) was constant and the calculated maximum velocity was 1.56 U/min; the thrombin Km value (0.011 nM) was affected by HARP concentrations. The Km (HARP) value was 0.085 nM, which is consistent with high affinity of HARP to OTR4120. Under the same conditions, initial velocity patterns for antithrombin-heparin were determined in the presence or in the absence of HARP. The antithrombin value Km (0.022 nM) was affected by HARP (0.077 nM). HARP exhibits efficacy equivalent to or greater than protamine. Interestingly, intraperitoneally administered HARP decreased the anticoagulant activity of heparin and of OTR4120 in mice. Taken together, these data provide the first evidence for a physiological role of HARP in the modulation of anticoagulant activity of heparin and heparin-like material.

  16. Dopamine transporter oligomerization: Impact of combining protomers with differential cocaine analog binding affinities

    PubMed Central

    Zhen, Juan; Antonio, Tamara; Cheng, Shu-Yuan; Ali, Solav; Jones, Kymry T.; Reith, Maarten E. A.

    2015-01-01

    Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [3H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious “mixing” experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among 5 pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [3H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer. PMID:25580950

  17. [[Preparation of boronate affinity sorbent and its extraction performance for benzoylurea pesticides

    PubMed

    Zhang, Yong; Mei, Meng; Liu, Yi; Yu, Jie; Huang, Xiaojia; Yuan, Dongxing

    2014-09-01

    A new boronate affinity monolithic material was prepared and used as the extraction medium of stir cake sorptive extraction (SCSE). The porous boronate affinity sorbent was prepared by in situ copolymerization of 3-acrylamidophenylboronic acid (APB) and divinyl benzene (DVB). To achieve optimum extraction performance for benzoylurea pesticides, several parameters, including desorption solvent, pH value, ionic strength in sample matrix, extraction and desorption times, were investigated in detail. At the same time, a simple, sensitive and environment friendly method for the determination of benzoylurea pesticides in water and juice samples was developed by the combination of SCSE-APBDVB with HPLC equipped with a diode array detector. Under the optimized experimental conditions, the limits of detection (S/N = 3) for target analytes were 0.055-0.11 μg/L in water and 0.095-0.31 μg/L in juice. The precision of the proposed method was evaluated in terms of intra- and inter-assay repeatability calculated as RSD, and it was found that the RSDs were all below 9.0%. The developed method was successfully applied to the determination of benzoylurea pesticide residues in water and juice samples and satisfactory recoveries of spiked target compounds were in the range of 75.6%-109%. The results well demonstrate that the new sorbent can extract benzoylurea pesticides effectively through multi-interactions including boron-nitrogen coordination, hydrogen-bond and hydrophobic interactions between sorbent and analytes. PMID:25752092

  18. Characterization of a high affinity cocaine binding site in rat brain

    SciTech Connect

    Calligaro, D.; Eldefrawi, M.

    1986-03-05

    Binding of (/sup 3/H)cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of (/sup 3/H)cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95/sup 0/C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of (/sup 3/H)cocaine (15 nM) was inhibited by increasing concentrations of Na/sup +/ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific (/sup 3/H)cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC/sub 50/ = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting (/sup 3/H)cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC/sub 50/ values below ..mu..M concentrations.

  19. Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases*

    PubMed Central

    Sørensen, Trine Holst; Cruys-Bagger, Nicolaj; Windahl, Michael Skovbo; Badino, Silke Flindt; Borch, Kim; Westh, Peter

    2015-01-01

    We measured hydrolytic rates of four purified cellulases in small increments of temperature (10–50 °C) and substrate loads (0–100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q10 values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases. PMID:26183777

  20. Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

    PubMed Central

    Inoue, Atsushi; Takagi, Yasuomi; Taira, Kazunari

    2004-01-01

    Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway—a cooperative pathway—in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst. PMID:15302920

  1. Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases.

    PubMed

    Sørensen, Trine Holst; Cruys-Bagger, Nicolaj; Windahl, Michael Skovbo; Badino, Silke Flindt; Borch, Kim; Westh, Peter

    2015-09-01

    We measured hydrolytic rates of four purified cellulases in small increments of temperature (10-50 °C) and substrate loads (0-100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q10 values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases. PMID:26183777

  2. Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives

    PubMed Central

    Rios, Pablo; Carter, Tom S.; Crump, Matthew P.; Lisbjerg, Micke; Pittelkow, Michael; Supekar, Nitin T.; Boons, Geert‐Jan

    2016-01-01

    Abstract The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water‐soluble carbohydrate receptors (“synthetic lectins”). Both systems show outstanding affinities for derivatives of N‐acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc‐β‐OMe with K a≈20 000 m −1, whereas the other one binds an O‐GlcNAcylated peptide with K a≈70 000 m −1. These values substantially exceed those usually measured for GlcNAc‐binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl–peptide complex can be explained by extra‐cavity interactions, raising the possibility of a family of complementary receptors for O‐GlcNAc in different contexts. PMID:26822115

  3. Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel.

    PubMed

    Beyaztaş, Serap; Arslan, Oktay

    2015-06-01

    A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150 kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60 °C and incubated for 60 min. These results indicated that the enzyme was heat stable. PMID:25089709

  4. Net charge and oxygen affinity of human hemoglobin are independent of hemoglobin concentration

    PubMed Central

    1978-01-01

    The dependence of net charge and oxygen affinity of human hemoglobin upon hemoglobin concentration was reinvestigated. In contrast to earlier reports from various laboratories, both functional properties of hemoglobin were found to be independent of hemoglobin concentration. Two findings indicate a concentration-independent net charge of carbonmonoxy hemoglobin at pH 6.6: (A) The pH value of a given carbonmonoty hemoglobin solution remains constant at 6.6 when the hemoglobin concentration is raised from 10 to 40 g/dl, indicating that there is no change in protonation of titratable groups of hemoglobin: (b) the net charge of carbonmonoxy hemoglobin as estimated from the Donnan distribution of 22Na+ shows no dependence on hemoglobin concentration in this concentration range. The oxygen affinity of human hemoglobin was determined from measurements of oxygen concentrations in equilibrated samples using a Lex-O2-Con apparatus (Lexington Instruments, Waltham, Mass.). P50 averaged 11.4 mm Hg at 37 degrees C, pH = 7.2, and ionic strength approximately 0.15. Neither P50 nor Hill's n showed any variation with hemoglobin concentrations increasing from 10 to 40 g/dl. PMID:32221

  5. Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration affinity membrane.

    PubMed

    Konovalova, Viktoriia; Guzikevich, Kateryna; Burban, Anatoliy; Kujawski, Wojciech; Jarzynka, Karolina; Kujawa, Joanna

    2016-11-01

    In order to prepare ultrafiltration membranes possessing biocatalytic properties, α-amylase has been immobilized on cellulose membranes. Enzyme immobilization was based on a covalent bonding between chitosan and a surface of cellulose membrane, followed by an attachment of Cibacron Blue F3G-A dye as affinity ligand. Various factors affecting the immobilization process, such as enzyme concentration, pH of modifying solution, zeta-potential of membrane surface, and stability of immobilized enzyme were studied. The applicability of immobilized α-amylase has been investigated in ultrafiltration processes. The immobilization of α-amylase on membrane surface allows to increase the value of mass transfer coefficient and to decrease the concentration polarization effect during ultrafiltration of starch solutions. The enzyme layer on the membrane surface prevents a rapid increase of starch concentration due to the amylase hydrolysis of starch in the boundary layer. The presented affinity immobilization technique allows also for the regeneration of membranes from inactivated enzyme. PMID:27516322

  6. Regulator of insulin receptor affinity in rat skeletal muscle sarcolemmal vesicles

    SciTech Connect

    Whitson, R.H.; Barnard, K.J.; Kaplan, S.A.; Itakura, K.

    1986-05-01

    Wheat germ agglutinin (WGA) affinity purification of detergent solubilized insulin receptors (IR) from rat skeletal muscle sarcolemmal vesicles resulted in an apparent increase in total insulin binding activity of greater than 5-fold, suggesting that an inhibitory component had been removed. This was verified when the flow-through fraction from the WGA column was dialyzed and added back to the partially purified receptor. The addition of a 100-fold dilution of the inhibitor preparation caused a 50% reduction in binding to trace amounts of /sup 125/I-insulin. Scatchard analysis revealed that the effect of the inhibitor was to decrease the affinity of the muscle IR. The inhibitor appeared to be tissue specific, inasmuch as the I/sub 50/'s for WGA-purified IR from rat fat and liver were 10 times the I/sub 50/ for muscle IR. The I/sub 50/ for insulin binding to intact IM-9 cells was 30 times the value for muscle IR. The inhibitor eluted in the void volume of Sephadex G-50 columns. Its activity was not destroyed by heating at 90/sup 0/C for 10 minutes, or by prolonged incubation with trypsin or dithiothreitol. The inhibitor described here may have a role in modulating insulin sensitivity in skeletal muscle.

  7. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  8. An affine point-set and line invariant algorithm for photo-identification of gray whales

    NASA Astrophysics Data System (ADS)

    Chandan, Chandan; Kehtarnavaz, Nasser; Hillman, Gilbert; Wursig, Bernd

    2004-05-01

    This paper presents an affine point-set and line invariant algorithm within a statistical framework, and its application to photo-identification of gray whales (Eschrichtius robustus). White patches (blotches) appearing on a gray whale's left and right flukes (the flattened broad paddle-like tail) constitute unique identifying features and have been used here for individual identification. The fluke area is extracted from a fluke image via the live-wire edge detection algorithm, followed by optimal thresholding of the fluke area to obtain the blotches. Affine point-set and line invariants of the blotch points are extracted based on three reference points, namely the left and right tips and the middle notch-like point on the fluke. A set of statistics is derived from the invariant values and used as the feature vector representing a database image. The database images are then ranked depending on the degree of similarity between a query and database feature vectors. The results show that the use of this algorithm leads to a reduction in the amount of manual search that is normally done by marine biologists.

  9. Polymer-ceramic Monolithic In-Needle Extraction (MINE) device: Preparation and examination of drug affinity.

    PubMed

    Pietrzyńska, Monika; Tomczak, Rafał; Jezierska, Katarzyna; Voelkel, Adam; Jampílek, Josef

    2016-11-01

    Polymer-ceramic materials were placed in the in-needle device. Polymer-ceramic Monolithic In-Needle Extraction (MINE) device is an extraction device used in sample preparation step but, on the other hand, it can be a tool for examination of interactions between potential antiresorptive drugs and bones. MINE device was used as tool for determination of bisphosphonate affinity to hydroxyapatite. Spectra of prepared materials containing different proportion of the ceramic part were performed with the use of Fourier transform infrared spectroscopy. The extraction of sodium risedronate as standard compound from simulated body fluids was carried out by pumping liquid samples through the MINE device. The amount of sodium risedronate in solutions was examined using UV-VIS spectroscopy. The sorption results of sodium risedronate obtained for monolithic materials containing different amount of hydroxyapatite were compared to the values determined for pure (bulk) hydroxyapatite. Sorption capacity for polymer-ceramic materials placed in the in-needle extraction device was about 0.39mg of sodium risedronate. The complete desorption process was carried out at the level over 95% using various eluents. The results of sorption-desorption experiments allow to deduce on the affinity of sodium risedronate to the ceramic part of sorbent (hydroxyapatite). PMID:27523998

  10. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    SciTech Connect

    Lach, E.; Trifilieff, A.; Landry, Y.; Gies, J.P. )

    1991-01-01

    The binding of the radiolabeled bombesin analogue ({sup 125}I-Tyr{sup 4})bombesin to guinea-pig lung membranes was investigated. Binding of ({sup 125}I-Tyr{sup 4})bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B{sub max} = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K{sub D} = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as ({sup 125}I-Tyr{sup 4})bombesin, neuromedin B and neuromedin C inhibited the binding of ({sup 125}I-Tyr{sup 4})bombesin in an order of potencies as follows: ({sup 125}I-Tyr{sup 4})bombesin {gt} bombesin {ge} neuromedin C {much gt} neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.

  11. A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

    PubMed

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2014-11-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

  12. Spatial distribution of diuron sorption affinity as affected by soil, terrain and management practices in an intensively managed apple orchard.

    PubMed

    Umali, Beng P; Oliver, Danielle P; Ostendorf, Bertram; Forrester, Sean; Chittleborough, David J; Hutson, John L; Kookana, Rai S

    2012-05-30

    We investigated how the sorption affinity of diuron (3'-(3,4-dichlorophenyl)-1,1-dimenthyl-urea), a moderately hydrophobic herbicide, is affected by soil properties, topography and management practices in an intensively managed orchard system. Soil-landscape analysis was carried out in an apple orchard which had a strong texture contrast soil and a landform with relief difference of 50 m. Diuron sorption (K(d)) affinity was successfully predicted (R(2)=0.79; p<0.001) using a mid-infrared - partial least squares model and calibrated against measured data using a conventional batch sorption technique. Soil and terrain properties explained 75% of the variance of diuron K(d) with TOC, pH(w), slope and WI as key variables. Mean diuron K(d) values were also significantly different (p<0.05) between alley and tree line and between the different management zones. Soil in the tree line generally had lower sorption capacity for diuron than soil in the alleys. Younger stands, which were found to have lower TOC than in the older stands, also had lower diuron K(d) values. In intensively managed orchards, sorption affinity of pesticides to soils was not only affected by soil properties and terrain attributes but also by management regime.

  13. Affinity covalent immobilization of glucoamylase onto ρ-benzoquinone-activated alginate beads: II. Enzyme immobilization and characterization.

    PubMed

    Eldin, M S Mohy; Seuror, E I; Nasr, M A; Tieama, H A

    2011-05-01

    A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K (m) values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V (m) value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.

  14. Synthesis and Binding Affinity of Novel Mono- and Bivalent Morphinan Ligands for κ, μ and δ Opioid Receptors

    PubMed Central

    Zhang, Bin; Zhang, Tangzhi; Sromek, Anna W.; Scrimale, Thomas; Bidlack, Jean M.

    2011-01-01

    A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors and their functional activities were determined at κ and μ receptors in [35S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (Ki values less than 1 nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity Ki values of 0.089 nM at the μ receptor and 0.073 nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists. PMID:21482470

  15. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  16. Quantitative Structure-Activity Relationship for High Affinity 5-HT1A Receptor Ligands Based on Norm Indexes.

    PubMed

    Jia, Qingzhu; Cui, Xue; Li, Lei; Wang, Qiang; Liu, Ying; Xia, Shuqian; Ma, Peisheng

    2015-12-24

    Arylpiperazine derivatives are promising 5-hydroxytryptamine (5-HT) receptor ligands which can inhibit serotonin reuptake effectively. In this work, some norm index descriptors were proposed and further utilized to develop a model for predicting 5-HT1A receptor affinity (pKi) of 88 arylpiperazine derivatives. Results showed that this new model could provide satisfactory predictions with the square of the correction coefficient (R(2)) of 0.8891 and the squared correlation coefficient of cross-validation (Q(2)) of 0.8082, respectively. In addition, the applicability domain of this model was validated by using the leverage approach and results which suggested potential large scale for further utilization of this model. The results of statistical values and validation tests demonstrated that our proposed norm index based model could be successfully applied for predicting the affinity 5-HT1A receptor ligands of arylpiperazine derivatives.

  17. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  18. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach.

    PubMed

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N-terminal sublibrary, and 0.77 and 269.13 for C-terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  19. Evaluation of phytochemical content, nutritional value and antioxidant activity of Phanji - Rivea hypocrateriformis (Desr.) Choisy leaf

    PubMed Central

    Borkar, Sneha D.; Naik, Raghavendra; Shukla, Vinay J.; Acharya, Rabinarayan

    2015-01-01

    Background: Rivea hypocrateriformis (Desr.) Choisy is known to be the source plant of Phanji, a classically delineated leafy vegetable which is till date used by some hill dwelling Kandha tribes of Odisha. Though it is in use since a long time, it is not yet evaluated for its nutritive value. Aim: The leaves of R. hypocrateriformis were evaluated for its nutritive value and antioxidant potential. Materials and Methods: The in vitro antioxidant properties of the leaf of R. hypocrateriformis were screened through 1,1-diphenyl-2-picrylhydrazyl (DPPH) and total antioxidant capacity. Phytochemicals, crude protein, fat, carbohydrate, energy value, and mineral content of the leaves of the plant were evaluated with standard procedures. Results: In phytochemical analysis, tannin, alkaloids, flavonoids, and carbohydrates were present in leaf powder of R. hypocrateriformis. Energy content was found to be highest (331.54 kcals/100 g). Carbohydrate, fat, protein, calcium, magnesium, phosphorous, and zinc were present in 57.63%, 2.66%, 19.27%, 0.99%, 0.34%, 0.32%, and 0.011%, respectively. The IC50 values of the extract and ascorbic acid were found to be 254 ± 5.29 μg/ml and 11.67 ± 0.58 μg/ml, respectively. Percentage scavenging of DPPH radical was found to rise with increasing concentration of the crude extract. Total antioxidant capacity of the extract was found to be 111.30 ± 0.003 mcg. Conclusion: The results of this study indicate that the leaves of R. hypocrateriformis contain secondary metabolites such as tannin and possess mild antioxidant properties. Nutritional analysis indicates the presence of energy in highest amount, carbohydrates, proteins, fats, calcium, phosphorous, zinc, and magnesium. PMID:27313417

  20. Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit. Results from an increased apparent affinity for the A2 subunit.

    PubMed

    Monaghan, M; Wakabayashi, H; Griffiths, A; Wintermute, J; Fay, P J

    2014-09-01

    Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay. PMID:24899227

  1. Engineered affinity proteins for tumour-targeting applications.

    PubMed

    Friedman, Mikaela; Ståhl, Stefan

    2009-05-01

    Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are overexpressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid tumours. In the present review, strategies are described for tumour targeting of solid tumours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody) molecule. PMID:19341363

  2. Improving affinity chromatography resin efficiency using semi-continuous chromatography.

    PubMed

    Mahajan, Ekta; George, Anupa; Wolk, Bradley

    2012-03-01

    Protein A affinity chromatography is widely used for purification of monoclonal antibodies (MAbs) from harvested cell culture fluid (HCCF). At the manufacturing scale, the HCCF is typically loaded on a single Protein A affinity chromatography column in cycles until all of the HCCF is processed. Protein A resin costs are significant, comprising a substantial portion of the raw material costs in MAb manufacturing. Cost can be reduced by operating the process continuously using multiple smaller columns to a higher binding capacity in lieu of one industrial scale column. In this study, a series of experiments were performed using three 1-ml Hi-Trap™ MabSelect SuRe™ columns on a modified ÄKTA™ system operated according to the three Column Periodic Counter Current Chromatography (3C PCC) principle. The columns were loaded individually at different times until the 70% breakthrough point was achieved. The HCCF with unbound protein from the column was then loaded onto the next column to capture the MAb, preventing any protein loss. At any given point, all three columns were in operation, either loading or washing, enabling a reduction in processing time. The product yield and quality were evaluated and compared with a batch process to determine the effect of using the three column continuous process. The continuous operation shows the potential to reduce both resin volume and buffer consumption by ∼40%, however the system hardware and the process is more complex than the batch process. Alternative methods using a single standard affinity column, such as recycling load effluent back to the tank or increasing residence time, were also evaluated to improve Protein A resin efficiency. These alternative methods showed similar cost benefits but required longer processing time. PMID:22265178

  3. The affinity of magnetic microspheres for Schistosoma eggs.

    PubMed

    Candido, Renata R F; Favero, Vivian; Duke, Mary; Karl, Stephan; Gutiérrez, Lucía; Woodward, Robert C; Graeff-Teixeira, Carlos; Jones, Malcolm K; St Pierre, Timothy G

    2015-01-01

    Schistosomiasis is a chronic parasitic disease of humans, with two species primarily causing the intestinal infection: Schistosoma mansoni and Schistosoma japonicum. Traditionally, diagnosis of schistosomiasis is achieved through direct visualisation of eggs in faeces using techniques that lack the sensitivity required to detect all infections, especially in areas of low endemicity. A recently developed method termed Helmintex™ is a very sensitive technique for detection of Schistosoma eggs and exhibits 100% sensitivity at 1.3 eggs per gram of faeces, enough to detect even low-level infections. The Helminthex™ method is based on the interaction of magnetic microspheres and schistosome eggs. Further understanding the underlying egg-microsphere interactions would enable a targeted optimisation of egg-particle binding and may thus enable a significant improvement of the Helmintex™ method and diagnostic sensitivity in areas with low infection rates. We investigated the magnetic properties of S. mansoni and S. japonicum eggs and their interactions with microspheres with different magnetic properties and surface functionalization. Eggs of both species exhibited higher binding affinity to the magnetic microspheres than the non-magnetic microspheres. Binding efficiency was further enhanced if the particles were coated with streptavidin. Schistosoma japonicum eggs bound more microspheres compared with S. mansoni. However, distinct differences within eggs of each species were also observed when the distribution of the number of microspheres bound per egg was modelled with double Poisson distributions. Using this approach, both S. japonicum and S. mansoni eggs fell into two groups, one having greater affinity for magnetic microspheres than the other, indicating that not all eggs of a species exhibit the same binding affinity. Our observations suggest that interaction between the microspheres and eggs is more likely to be related to surface charge-based electrostatic

  4. Computational study of coagulation factor VIIa's affinity for phospholipid membranes.

    PubMed

    Taboureau, Olivier; Olsen, Ole Hvilsted

    2007-02-01

    The interaction between the gamma-carboxyglutamic acid-rich domain of coagulation factor VIIa (FVIIa), a vitamin-K-dependent enzyme, and phospholipid membranes plays a major role in initiation of blood coagulation. However, despite a high sequence and structural similarity to the Gla domain of other vitamin-K-dependent enzymes with a high membrane affinity, its affinity for negatively charged phospholipids is poor. A few amino acid differences are responsible for this observation. Based on the X-ray structure of lysophosphatidylserine (lysoPS) bound to the Gla domain of bovine prothrombin (Prth), models of the Gla domain of wildtype FVIIa and mutated FVIIa Gla domains in complex with lysoPS were built. Molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on the complexes were applied to investigate the significant difference in the binding affinity. The MD simulation approach provides a structural and dynamic support to the role of P10Q and K32E mutations in the improvement of the membrane contact. Hence, rotation of the Gly11 main chain generated during the MD simulation results in a hydrogen bond with Q10 side chain as well as the appearance of a hydrogen bond between E32 and Q10 forcing the loop harbouring Arg9 and Arg15 to shrink and thereby enhances the accessibility of the phospholipids to the calcium ions. Furthermore, the application of the SMD simulation method to dissociate C6-lysoPS from a series of Gla domain models exhibits a ranking of the rupture force that can be useful in the interpretation of the PS interaction with Gla domains. Finally, adiabatic mapping of Gla6 residue in FVIIa with or without insertion of Tyr4 confirms the critical role of the insertion on the conformation of the side chain Gla6 in FVIIa and the corresponding Gla7 in Prth. PMID:17131117

  5. The affinity of magnetic microspheres for Schistosoma eggs.

    PubMed

    Candido, Renata R F; Favero, Vivian; Duke, Mary; Karl, Stephan; Gutiérrez, Lucía; Woodward, Robert C; Graeff-Teixeira, Carlos; Jones, Malcolm K; St Pierre, Timothy G

    2015-01-01

    Schistosomiasis is a chronic parasitic disease of humans, with two species primarily causing the intestinal infection: Schistosoma mansoni and Schistosoma japonicum. Traditionally, diagnosis of schistosomiasis is achieved through direct visualisation of eggs in faeces using techniques that lack the sensitivity required to detect all infections, especially in areas of low endemicity. A recently developed method termed Helmintex™ is a very sensitive technique for detection of Schistosoma eggs and exhibits 100% sensitivity at 1.3 eggs per gram of faeces, enough to detect even low-level infections. The Helminthex™ method is based on the interaction of magnetic microspheres and schistosome eggs. Further understanding the underlying egg-microsphere interactions would enable a targeted optimisation of egg-particle binding and may thus enable a significant improvement of the Helmintex™ method and diagnostic sensitivity in areas with low infection rates. We investigated the magnetic properties of S. mansoni and S. japonicum eggs and their interactions with microspheres with different magnetic properties and surface functionalization. Eggs of both species exhibited higher binding affinity to the magnetic microspheres than the non-magnetic microspheres. Binding efficiency was further enhanced if the particles were coated with streptavidin. Schistosoma japonicum eggs bound more microspheres compared with S. mansoni. However, distinct differences within eggs of each species were also observed when the distribution of the number of microspheres bound per egg was modelled with double Poisson distributions. Using this approach, both S. japonicum and S. mansoni eggs fell into two groups, one having greater affinity for magnetic microspheres than the other, indicating that not all eggs of a species exhibit the same binding affinity. Our observations suggest that interaction between the microspheres and eggs is more likely to be related to surface charge-based electrostatic

  6. Novel phosphorus-containing cyclodextrin polymers and their affinity for calcium cations and hydroxyapatite.

    PubMed

    Wintgens, Véronique; Dalmas, Florent; Sébille, Bernard; Amiel, Catherine

    2013-10-15

    Novel phosphorous-containing β-cyclodextrin (βCD) polymers (CDP) were synthesized easily under "green chemistry" conditions. A simple polycondensation between the hydroxyl groups of βCD and non-toxic sodium trimetaphosphate (STMP) under basic conditions led to soluble, non-reticulated CDPs with molecular weights (Mw) higher than 10(4) g mol(-1), the actual value depending on the NaOH:βCD and STMP:βCD weight ratios. The presence of both βCD and phosphate groups in the polymer allows for strong interactions with amphiphilic probes, such as 1-adamantyl acetic acid, or with divalent cations, such as Ca(2+), whose strengths were characterized by isothermal titration microcalorimetry. The obtained phosphated compounds also display high affinity towards hydroxyapatite (HA), leading to HA nanoparticles that could easily be recovered by CDPs, as demonstrated by transmission electron microscopy and quantitative determination of the total amount of phosphated molecules fixed on HA. PMID:23987426

  7. Calculation of the electron affinities of the chromophores involved in photosynthesis

    SciTech Connect

    Cory, M.G.; Zerner, M.C.

    1996-05-01

    We examine the electron affinities (EA) of the postulated electron acceptor chromophores of the photosynthetic reaction center in R. Viridis. We estimate a difference of EA`s between bacteriochlorophyll-b and bacteriopheophytin-b of 0.19eV, in excellent agreement with the experimental value reported at 0.20 eV. We estimate this difference in situ at 0.42 eV, compared to an experimental estimate of 0.34 eV. These results support those of Thompson and Zerner that in the absence of specific interactions between BChl-L-side and the environment, electron transfer to BChl-L-side might not be directly involved in the overall electron transfer process. 42 refs., 2 figs., 3 tabs.

  8. Low energy Mott polarimetry of electrons from negative electron affinity photocathodes

    SciTech Connect

    Ciccacci, F.; De Rossi, S.; Campbell, D.M.

    1995-08-01

    We present data on the spin polarization {ital P} and quantum yield {ital Y} of electrons photoemitted from negative electron affinity semiconductors, including GaAs(100), GaAsP(100) alloy, and strained GaAs layer epitaxially grown on a GaAsP(100) buffer. Near photothreshold the following values for {ital P}({ital Y}) are, respectively, obtained: 26% (2.5{times}10{sup {minus}2}), 40% (1{times}10{sup {minus}3}), and 60% (1.5{times}10{sup {minus}4}). We describe in detail the apparatus used containing a low energy (10--25 keV) Mott polarimeter. The system, completely fitted in a small volume ({similar_to}10{sup 4} cm{sup 3}) ultrahigh vacuum chamber, is intended as a test facility for characterizing candidate photocathode materials for spin polarized electron sources. {copyright} {ital 1995} {ital American} {ital Institute} {ital of} {ital Physics}.

  9. Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

    PubMed

    Mulcahy, P; Griffin, T; O'Carra, P

    1997-02-01

    The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata). PMID:9116492

  10. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  11. High affinity of water-soluble cryptophanes for cesium cations.

    PubMed

    Brotin, Thierry; Montserret, Roland; Bouchet, Aude; Cavagnat, Dominique; Linares, Mathieu; Buffeteau, Thierry

    2012-01-20

    Exceptionally high affinity for cesium cations was achieved in aqueous solution using two enantiopure cryptophanes. Complexation of cesium was evidenced by (133)Cs NMR spectroscopy and by electronic circular dichroism (ECD). Binding constants as high as 6 × 10(9) M(-1) have been measured by isothermal titration calorimetry (ITC). Very strong complexation of rubidium cations (K ~10(6) M(-1)) has also been measured. Chiral hosts allowed the detection of the two cations at low concentrations (μM) using ECD.

  12. Friction and plasticity between self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Luan, Binquan; Robbins, Mark; Harrison, Judith

    2006-03-01

    Simulations are used to study the contact area and adhesion between two amorphous solids with self-affine fractal surfaces, and the results are compared to continuum calculations. The friction between non-adhesive surfaces is proportional to load, but the coefficient of friction increases with roughness. The friction is much higher than expected for elasticallly deforming surfaces,^* and substantial plastic deformation is observed. Indeed, friction forces for different surface roughness collapse when plotted against the number of plastic rearrangements per unit sliding distance. Including adhesion leads to an increase in both friction and plasticity. ^* M. H. Müser, L. Wenning, and M. O. Robbins, Phys. Rev. Lett. 86, 1295 (2001).

  13. Nine switch-affine neurons suffice for Turing universality.

    PubMed

    Siegelmann, H T.; Margenstern, M

    1999-06-01

    In a previous work Pollack showed that a particular type of heterogeneous processor network is Turing universal. Siegelmann and Sontag (1991) showed the universality of homogeneous networks of first-order neurons having piecewise-linear activation functions. Their result was generalized by Kilian and Siegelmann (1996) to include various sigmoidal activation functions. Here we focus on a type of high-order neurons called switch-affine neurons, with piecewise-linear activation functions, and prove that nine such neurons suffice for simulating universal Turing machines. PMID:12662670

  14. An affine projection algorithm using grouping selection of input vectors

    NASA Astrophysics Data System (ADS)

    Shin, JaeWook; Kong, NamWoong; Park, PooGyeon

    2011-10-01

    This paper present an affine projection algorithm (APA) using grouping selection of input vectors. To improve the performance of conventional APA, the proposed algorithm adjusts the number of the input vectors using two procedures: grouping procedure and selection procedure. In grouping procedure, the some input vectors that have overlapping information for update is grouped using normalized inner product. Then, few input vectors that have enough information for for coefficient update is selected using steady-state mean square error (MSE) in selection procedure. Finally, the filter coefficients update using selected input vectors. The experimental results show that the proposed algorithm has small steady-state estimation errors comparing with the existing algorithms.

  15. Modified gravity in three dimensional metric-affine scenarios

    NASA Astrophysics Data System (ADS)

    Bambi, Cosimo; Ghasemi-Nodehi, M.; Rubiera-Garcia, D.

    2015-08-01

    We consider metric-affine scenarios where a modified gravitational action is sourced by electrovacuum fields in a three dimensional space-time. We first study the case of f (R ) theories, finding deviations near the center as compared to the solutions of general relativity. We then consider Born-Infeld gravity, which has raised a lot of interest in the last few years regarding its applications in astrophysics and cosmology, and show that new features always arise at a finite distance from the center. Several properties of the resulting space-times, in particular in presence of a cosmological constant term, are discussed.

  16. Calcium affinity of human α-actinin 1.

    PubMed

    Backman, Lars

    2015-01-01

    Due to alternative splicing, the human ACTN1 gene codes for three different transcripts of α-actinin; one isoform that is expressed only in the brain and two with a more general expression pattern. The sequence difference is located to the C-terminal domains and the EF-hand motifs. Therefore, any functional or structural distinction should involve this part of the protein. To investigate this further, the calcium affinities of these three isoforms of α-actinin 1 have been determined by isothermal calorimetry. PMID:26020004

  17. Kinetic analysis of drug-protein interactions by affinity chromatography.

    PubMed

    Bi, Cong; Beeram, Sandya; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-10-01

    Information on the kinetics of drug-protein interactions is of crucial importance in drug discovery and development. Several methods based on affinity chromatography have been developed in recent years to examine the association and dissociation rates of these processes. These techniques include band-broadening measurements, the peak decay method, peak fitting methods, the split-peak method, and free fraction analysis. This review will examine the general principles and applications of these approaches and discuss their use in the characterization, screening and analysis of drug-protein interactions in the body. PMID:26724332

  18. Enrichment of Phosphopeptides via Immobilized Metal Affinity Chromatography.

    PubMed

    Swaney, Danielle L; Villén, Judit

    2016-03-01

    Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved. PMID:26933247

  19. Analyzing a kinetic titration series using affinity biosensors.

    PubMed

    Karlsson, Robert; Katsamba, Phinikoula S; Nordin, Helena; Pol, Ewa; Myszka, David G

    2006-02-01

    The classical method of measuring binding constants with affinity-based biosensors involves testing several analyte concentrations over the same ligand surface and regenerating the surface between binding cycles. Here we describe an alternative approach to collecting kinetic binding data, which we call "kinetic titration." This method involves sequentially injecting an analyte concentration series without any regeneration steps. Through a combination of simulation and experimentation, we show that this method can be as robust as the classical method of analysis. In addition, kinetic titrations can be more efficient than the conventional data collection method and allow us to fully characterize analyte binding to ligand surfaces that are difficult to regenerate.

  20. Representations of affine superalgebras and mock theta functions. III

    NASA Astrophysics Data System (ADS)

    Kac, V. G.; Wakimoto, M.

    2016-08-01

    We study modular invariance of normalized supercharacters of tame integrable modules over an affine Lie superalgebra, associated to an arbitrary basic Lie superalgebra g. For this we develop a several step modification process of multivariable mock theta functions, where at each step a Zwegers' type 'modifier' is used. We show that the span of the resulting modified normalized supercharacters is \\operatorname{SL}_2( Z)-invariant, with the transformation matrix equal, in the case the Killing form on g is non-degenerate, to that for the basic defect 0 subalgebra g^! of g, orthogonal to a maximal isotropic set of roots of g.

  1. [Progresses in screening active compounds from herbal medicine by affinity chromatography].

    PubMed

    Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

    2015-03-01

    Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening. PMID:26226740

  2. Lactam based 7-amino suberoylamide hydroxamic acids as potent HDAC inhibitors.

    PubMed

    Taddei, Maurizio; Cini, Elena; Giannotti, Luca; Giannini, Giuseppe; Battistuzzi, Gianfranco; Vignola, Davide; Vesci, Loredana; Cabri, Walter

    2014-01-01

    A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7 of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data compared with the corresponding linear products, without substituent in position 7. In general, this modification provided an effective reinforcement of in vitro activity. While the lactam size or the CO/NH group orientation did not strongly influence the inhibition, the contemporary modification of the suberoylamide fragment gave vary active variants in the lactam series, with compound 28 (ST8078AA1) that showed IC50 values between 2 and 10nM against all Class I HDAC isoforms, demonstrating it to be a large spectrum pan-inhibitor. This strong affinity with HDAC was also confirmed by the value of IC50=0.5μM against H460 cells, ranking 28 as one of the most potent HDAC inhibitors described so far. PMID:24345446

  3. Search for novel histone deacetylase inhibitors. Part II: design and synthesis of novel isoferulic acid derivatives.

    PubMed

    Lu, Wen; Wang, Fang; Zhang, Tao; Dong, Jinyun; Gao, Hongping; Su, Ping; Shi, Yaling; Zhang, Jie

    2014-05-01

    Previously, we described the discovery of potent ferulic acid-based histone deacetylase inhibitors (HDACIs) with halogeno-acetanilide as novel surface recognition moiety (SRM). In order to improve the affinity and activity of these HDACIs, twenty seven isoferulic acid derivatives were described herein. The majority of title compounds displayed potent HDAC inhibitory activity. In particular, IF5 and IF6 exhibited significant enzymatic inhibitory activities, with IC50 values of 0.73 ± 0.08 and 0.57 ± 0.16 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against human cancer cells. Especially, IF6 displayed promising profile as an antitumor candidate with IC50 value of 3.91 ± 0.97 μM against HeLa cells. The results indicated that these isoferulic acid derivatives could serve as promising lead compounds for further optimization. PMID:24702857

  4. Molecular Docking Study on Galantamine Derivatives as Cholinesterase Inhibitors.

    PubMed

    Atanasova, Mariyana; Yordanov, Nikola; Dimitrov, Ivan; Berkov, Strahil; Doytchinova, Irini

    2015-06-01

    A training set of 22 synthetic galantamine derivatives binding to acetylcholinesterase was docked by GOLD and the protocol was optimized in terms of scoring function, rigidity/flexibility of the binding site, presence/absence of a water molecule inside and radius of the binding site. A moderate correlation was found between the affinities of compounds expressed as pIC50 values and their docking scores. The optimized docking protocol was validated by an external test set of 11 natural galantamine derivatives and the correlation coefficient between the docking scores and the pIC50 values was 0.800. The derived relationship was used to analyze the interactions between galantamine derivatives and AChE. PMID:27490385

  5. A new method of quantitative affinity chromatography and its application to the study of myosin.

    PubMed Central

    Bottomley, R C; Storer, A C; Trayer, I P

    1976-01-01

    A new method of quantifying the interactions between two or three components of an interacting system, one of which is insoluble, is described. The method differs from those previously applied to affinity chromatography systems in that it does not require that elution volumes be measured, but is instead dependent on measurements of the quantity of affinity-bound material. Theoretical expressions are derived for systems in which the acceptor is immobilized. Examples presented to illustrate the validity of the theory are of the latter type and are from studies on the myosin-adenosine nucleotide-PPi system. With Sepharose-myosin columns (myosin covalently coupled to CNBr-activated Sepharose) a dissociation constant of 1.8 muM for ATP4- was found. Data were also obtained under conditions that closely approximate to those found in vivo, i.e. on columns packed with a slurry of Sephadex G-50 and precipitated myosin filaments formed at low ionic strength. The binding of MgATP2-, MgADP-, ATP4- and MgPPi2- to "filamentous" myosin in both two- (myosin and nucleotide) and three- (myosin, nucleotide and PPi) component systems at different temperatures was studied and the dissociation constants obtained agreed well with previously published values. Except for the binding of ATP4- to filamentous myosin at 4 degrees when 85% of the protein was interacting with the nucleotide, much lower values for the number of available sites occupied by the nucleotides were as a routine found in this system. Although this apparent discrepancy is difficult to explain, it is not an anomaly of the theoretical approach and may reflect the present state of understanding of the myosin system. PMID:1008824

  6. Mn-citrate and Mn-HIDA: intermediate-affinity chelates for manganese-enhanced MRI.

    PubMed

    Seo, Yoshiteru; Satoh, Keitaro; Morita, Hironobu; Takamata, Akira; Watanabe, Kazuto; Ogino, Takashi; Hasebe, Tooru; Murakami, Masataka

    2013-01-01

    In this study we investigated two manganese chelates in order to improve the image enhancement of manganese-enhanced MRI and decrease the toxicity of free manganese ions. Since both MnCl₂ and a low-affinity chelate were associated with a slow continuous decrease of cardiac functions, we investigated intermediate-affinity chelates: manganese N-(2-hydroxyethyl)iminodiacetic acid (Mn-HIDA) and Mn-citrate. The T₁ relaxivity values for Mn-citrate (4.4 m m⁻¹ s⁻¹) and Mn-HIDA (3.3 m m⁻¹ s⁻¹) in artificial cerebrospinal fluid (CSF) were almost constant in a concentration range from 0.5 to 5 m m at 37 °C and 4.7 T. In human plasma, the relaxivity values increased when the concentrations of these Mn chelates were decreased, suggesting the presence of free Mn²⁺ bound with serum albumin. Mn-HIDA and Mn-citrate demonstrated a tendency for better contractility when employed with an isolated perfused frog heart, compared with MnCl₂. Only minimal changes were demonstrated after a venous infusion of 100 m m Mn-citrate or Mn-HIDA (8.3 µmol kg⁻¹ min⁻¹) in rats and a constant heart rate, arterial pressure and sympathetic nerve activity were maintained, even after breaking the blood-brain barrier (BBB). Mn-citrate and Mn-HIDA could not cross the intact BBB and appeared in the CSF, and then diffused into the brain parenchyma through the ependymal layer. The responses in the supraoptic nucleus induced by the hypertonic stimulation were detectable. Therefore, Mn-citrate and Mn-HIDA appear to be better choices for maintaining the vital conditions of experimental animals, and they may improve the reproducibility of manganese-enhanced MRI of the small nuclei in the hypothalamus and thalamus. PMID:23281286

  7. Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters.

    PubMed

    Pham-Huu, Duy-Phong; Deschamps, Jeffrey R; Liu, Shanghao; Madras, Bertha K; Meltzer, Peter C

    2007-01-15

    Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers. PMID:17070057

  8. Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters.

    PubMed

    Pham-Huu, Duy-Phong; Deschamps, Jeffrey R; Liu, Shanghao; Madras, Bertha K; Meltzer, Peter C

    2007-01-15

    Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers.

  9. Sequential class switching is required for the generation of high affinity IgE antibodies

    PubMed Central

    Xiong, Huizhong; Dolpady, Jayashree; Wabl, Matthias; Curotto de Lafaille, Maria A.

    2012-01-01

    IgE antibodies with high affinity for their antigens can be stably cross-linked at low concentrations by trace amounts of antigen, whereas IgE antibodies with low affinity bind their antigens weakly. In this study, we find that there are two distinct pathways to generate high and low affinity IgE. High affinity IgE is generated through sequential class switching (μ→γ→ε) in which an intermediary IgG phase is necessary for the affinity maturation of the IgE response, where the IgE inherits somatic hypermutations and high affinity from the IgG1 phase. In contrast, low affinity IgE is generated through direct class switching (μ→ε) and is much less mutated. Mice deficient in IgG1 production cannot produce high affinity IgE, even after repeated immunizations. We demonstrate that a small amount of high affinity IgE can cause anaphylaxis and is pathogenic. Low affinity IgE competes with high affinity IgE for binding to Fcε receptors and prevents anaphylaxis and is thus beneficial. PMID:22249450

  10. Challenges and recent advances in affinity purification of tag-free proteins.

    PubMed

    Guan, Dongli; Chen, Zhilei

    2014-07-01

    There is currently no generic, simple, lowcost method for affinity chromatographic purification of proteins in which the purified product is free of appended tags. Existing approaches for the purification of tagless proteins fall into two broad categories: (1) direct affinity-based capture of tag-free proteins that utilize affinity ligands specific to the target protein or class of target protein, and (2) removal of an appended affinity tag following tag-mediated protein capture. This paper reviews current state-of-the-art approaches for tagless protein purification in both categories, including specific examples of affinity ligands used for the capture of different classes of proteins and cleavage systems for affinity tag removal following chromatographic capture. A particular focus of this review is on recent developments in affinity tag removal systems utilizing split inteins. PMID:24658742

  11. Bidentate Zinc Chelators for α-Carbonic Anhydrases that Produce a Trigonal Bipyramidal Coordination Geometry

    PubMed Central

    Wischeler, Johannes Schulze; Innocenti, Alessio; Vullo, Daniela; Agrawal, Arpita; Cohen, Seth M.; Heine, Andreas; Supuran, Claudiu T.; Klebe, Gerhard

    2010-01-01

    A series of new zinc binding groups (ZBGs) has been evaluated kinetically on 13 carbonic anhydrase (CA) isoforms. The fragments show affinity for all isoforms with IC50 values in the range of 2–11 µm. The crystal structure of hCA II in complex with one such fragment reveals a bidentate binding mode with a trigonal-bipyramidal coordination geometry at the Zn2+ center. The fragment also interacts with Thr199 and Thr200 through hydrogen bonding and participates in a water network. Further development of this ZBG should increase the binding affinity leading to a structurally distinct and promising class of CA inhibitors. PMID:20629007

  12. [Racemation of the benzodiazepines camazepam and ketazolam and receptor binding of enantiomers].

    PubMed

    Blaschke, G; Kley, H; Müller, W E

    1986-06-01

    The chiral benzodiazepines camazepam (1) and ketazolam (2) were resolved into the enantiomers by chromatography on the optically active adsorbent poly[(S)-N-(1-cyclohexylethyl)-methacrylamide] and fractional crystallisation or repeated chromatography, respectively. The IC50 values of the isomers and of the racemates of both compounds were determined by displacement of radioactively labelled 3H-flunitrazepam and 3H-propyl-beta-carboline-carboxylate from their specific binding sites. (+)-Camazepam exhibits 14fold higher affinity compared to the (-)-enantiomer. In contrast only slight differences in the receptor affinity are observed with the ketazolam enantiomers.

  13. Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity.

    PubMed

    Krummey, Scott M; Martinez, Ryan J; Andargachew, Rakieb; Liu, Danya; Wagener, Maylene; Kohlmeier, Jacob E; Evavold, Brian D; Larsen, Christian P; Ford, Mandy L

    2016-03-15

    Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice, but not in high affinity-primed mice. Mechanistically, low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge, low affinity-primed CD45RB(hi) cells became CD45RB(lo), demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge. PMID:26864034

  14. Affinity sensor using 3-aminophenylboronic acid for bacteria detection.

    PubMed

    Wannapob, Rodtichoti; Kanatharana, Proespichaya; Limbut, Warakorn; Numnuam, Apon; Asawatreratanakul, Punnee; Thammakhet, Chongdee; Thavarungkul, Panote

    2010-10-15

    Boronic acid that can reversibly bind to diols was used to detect bacteria through its affinity binding reaction with diol-groups on bacterial cell walls. 3-aminophenylboronic acid (3-APBA) was immobilized on a gold electrode via a self-assembled monolayer. The change in capacitance of the sensing surface caused by the binding between 3-APBA and bacteria in a flow system was detected by a potentiostatic step method. Under optimal conditions the linear range of 1.5×10(2)-1.5×10(6) CFU ml(-1) and the detection limit of 1.0×10(2) CFU ml(-1) was obtained. The sensing surface can be regenerated and reused up to 58 times. The method was used for the analysis of bacteria in several types of water, i.e., bottled, well, tap, reservoir and wastewater. Compared with the standard plate count method, the results were within one standard deviation of each other. The proposed method can save both time and cost of analysis. The electrode modified with 3-APBA would also be applicable to the detection of other cis-diol-containing analytes. The concept could be extended to other chemoselective ligands, offering less expensive and more robust affinity sensors for a wide range of compounds.

  15. Influence of affinity on antibody determination in microtiter ELISA systems

    SciTech Connect

    Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

    1986-03-01

    Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of /sup 125/I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of /sup 125/I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations.

  16. Telonemia, a new protist phylum with affinity to chromist lineages.

    PubMed

    Shalchian-Tabrizi, K; Eikrem, W; Klaveness, D; Vaulot, D; Minge, M A; Le Gall, F; Romari, K; Throndsen, J; Botnen, A; Massana, R; Thomsen, H A; Jakobsen, K S

    2006-07-22

    Recent molecular investigations of marine samples taken from different environments, including tropical, temperate and polar areas, as well as deep thermal vents, have revealed an unexpectedly high diversity of protists, some of them forming deep-branching clades within important lineages, such as the alveolates and heterokonts. Using the same approach on coastal samples, we have identified a novel group of protist small subunit (SSU) rDNA sequences that do not correspond to any phylogenetic group previously identified. Comparison with other sequences obtained from cultures of heterotrophic protists showed that the environmental sequences grouped together with Telonema, a genus known since 1913 but of uncertain taxonomic affinity. Phylogenetic analyses using four genes (SSU, Hsp90, alpha-tubulin and beta-tubulin), and accounting for gamma- and covarion-distributed substitution rates, revealed Telonema as a distinct group of species branching off close to chromist lineages. Consistent with these gene trees, Telonema possesses ultrastructures revealing both the distinctness of the group and the evolutionary affinity to chromist groups. Altogether, the data suggest that Telonema constitutes a new eukaryotic phylum, here defined as Telonemia, possibly representing a key clade for the understanding of the early evolution of bikont protist groups, such as the proposed chromalveolate supergroup. PMID:16790418

  17. Electron affinity of cubic boron nitride terminated with vanadium oxide

    NASA Astrophysics Data System (ADS)

    Yang, Yu; Sun, Tianyin; Shammas, Joseph; Kaur, Manpuneet; Hao, Mei; Nemanich, Robert J.

    2015-10-01

    A thermally stable negative electron affinity (NEA) for a cubic boron nitride (c-BN) surface with vanadium-oxide-termination is achieved, and its electronic structure was analyzed with in-situ photoelectron spectroscopy. The c-BN films were prepared by electron cyclotron resonance plasma-enhanced chemical vapor deposition employing BF3 and N2 as precursors. Vanadium layers of ˜0.1 and 0.5 nm thickness were deposited on the c-BN surface in an electron beam deposition system. Oxidation of the metal layer was achieved by an oxygen plasma treatment. After 650 °C thermal annealing, the vanadium oxide on the c-BN surface was determined to be VO2, and the surfaces were found to be thermally stable, exhibiting an NEA. In comparison, the oxygen-terminated c-BN surface, where B2O3 was detected, showed a positive electron affinity of ˜1.2 eV. The B2O3 evidently acts as a negatively charged layer introducing a surface dipole directed into the c-BN. Through the interaction of VO2 with the B2O3 layer, a B-O-V layer structure would contribute a dipole between the O and V layers with the positive side facing vacuum. The lower enthalpy of formation for B2O3 is favorable for the formation of the B-O-V layer structure, which provides a thermally stable surface dipole and an NEA surface.

  18. Reflection symmetry detection using locally affine invariant edge correspondence.

    PubMed

    Wang, Zhaozhong; Tang, Zesheng; Zhang, Xiao

    2015-04-01

    Reflection symmetry detection receives increasing attentions in recent years. The state-of-the-art algorithms mainly use the matching of intensity-based features (such as the SIFT) within a single image to find symmetry axes. This paper proposes a novel approach by establishing the correspondence of locally affine invariant edge-based features, which are superior to the intensity based in the aspects that it is insensitive to illumination variations, and applicable to textureless objects. The locally affine invariance is achieved by simple linear algebra for efficient and robust computations, making the algorithm suitable for detections under object distortions like perspective projection. Commonly used edge detectors and a voting process are, respectively, used before and after the edge description and matching steps to form a complete reflection detection pipeline. Experiments are performed using synthetic and real-world images with both multiple and single reflection symmetry axis. The test results are compared with existing algorithms to validate the proposed method. PMID:25608306

  19. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  20. Compensating Enthalpic and Entropic Changes Hinder Binding Affinity Optimization

    SciTech Connect

    Lafont,V.; Armstrong, A.; Ohtaka, H.; Kiso, Y.; Amzel, L.; Freire, E.

    2007-01-01

    A common strategy to improve the potency of drug candidates is to introduce chemical functionalities, like hydrogen bond donors or acceptors, at positions where they are able to establish strong interactions with the target. However, it is often observed that the added functionalities do not necessarily improve potency even if they form strong hydrogen bonds. Here, we explore the thermodynamic and structural basis for those observations. KNI-10033 is a potent experimental HIV-1 protease inhibitor with picomolar affinity against the wild-type enzyme (Kd = 13 pm). The potency of the inhibitor is the result of favorable enthalpic (?H = -8.2 kcal/mol) and entropic (-T?S = -6.7 kcal/mol) interactions. The replacement of the thioether group in KNI-10033 by a sulfonyl group (KNI-10075) results in a strong hydrogen bond with the amide of Asp 30B of the HIV-1 protease. This additional hydrogen bond improves the binding enthalpy by 3.9 kcal/mol; however, the enthalpy gain is completely compensated by an entropy loss, resulting in no affinity change. Crystallographic and thermodynamic analysis of the inhibitor/protease complexes indicates that the entropy losses are due to a combination of conformational and solvation effects. These results provide a set of practical guidelines aimed at overcoming enthalpy/entropy compensation and improve binding potency.